Side population (SP) refers to a group of cells, which is capable to efflux Hoechst 33342, a DNA-binding dye. SP cells exist both in normal and tumor tissues. SP abundance is used as an indicator for disease prognosis and drug screening in some studies. Our study concentrates on the factors influencing SP analysis.
DOI:10.21276/ijlssr.2016.2.4.2
neoplastic progression through the action of viral oncoproteins, mainly E6 and E7.Cervical cancer remains the second
most common cancer in women worldwide with India as a major contributor to global burden with an annual incidence of
132,000 new cases and mortality rate of 74,000 deaths annually. In this study turmeric, neem, tulasi and ginger were
selected as natural anticancer drugs. The objective of the study was to analyze the anticancer property of turmeric
(Curcuma longa), neem (Azadirachta indica), tulasi (Occimum sanctum) and ginger (Zingiber officinale) on HeLa cells.
Turmeric, neem, tulasi and ginger capsules (Himalaya’s Company) were used and aqueous and methanolic extracts of the
turmeric, neem, tulasi and ginger were obtained using a soxhlet extraction. To check the efficacy of these drug MTT assay
was performed, that determines % viability and/or cytotoxicity. IC50 of aqueous turmeric, neem, tulasi and ginger extracts
in case of HeLa cells were 17.8, 22, 79.4, 27.86 respectively and in case of methanolic turmeric, neem, tulasi and ginger
extracts 17, 7.35, 75.24 and 16.1 respectively. To confirm apoptosis as the sole reason behind cell death
immunofluorescence based apoptosis assay was performed using TALI image based cytometer. The study has led to
postulate hypothesis that natural drugs e.g. turmeric, neem, tulasi and ginger are potent anti-cancer compound that are
capable of inhibiting the growth of immortal cells by apoptosis. Key-words- Cervical cancer, Human papillomavirus (HPV), Oncoproteins E6 and E7, Natural compounds, HeLa cell
line (adherent), Cell viability and MTT assay, Apoptosis assay
1) The study investigated the effects of the tyrosine kinase inhibitors crizotinib and dasatinib on canine osteosarcoma cell lines. It found that dasatinib more effectively suppressed cell viability and inhibited hepatocyte growth factor-induced invasion and migration compared to crizotinib.
2) Exogenous hepatocyte growth factor increased invasion, migration, and matrix metalloproteinase production in the osteosarcoma cell lines.
3) Preliminary results showed that four dogs with osteosarcoma treated with adjuvant dasatinib therapy experienced an apparent clinical benefit.
This document discusses several in vitro methods for assessing the cytotoxicity of chemotherapeutic drugs, including assays using brine shrimp, MTT, sulforhodamine B, trypan blue dye, and acridine orange/ethidium bromide staining. It describes maintaining cell lines from cervical carcinoma and breast adenocarcinoma in culture, assessing cell viability, and preserving cells in liquid nitrogen. Specific methods are provided for assays including brine shrimp lethality, MTT, sulforhodamine B, trypan blue dye exclusion, acridine orange/ethidium bromide staining, and DNA fragmentation to evaluate the cytotoxic effects of test compounds on cultured malignant cell lines.
This document describes a screen of 50,000 compounds to identify inhibitors of hepatocyte growth factor (HGF)-induced epithelial scattering. Several structurally distinct compounds were identified that had not previously been reported to have biological activity. Further analysis revealed that many of the compounds broadly inhibit cancer cell proliferation by arresting cell division in G2/M phase with minimal induction of apoptosis. This effect is consistent with microtubule-targeting agents. Biochemical assays showed that several compounds directly inhibit microtubule polymerization. Some pharmacokinetic properties of the compounds were also assessed.
This study investigated the effects of dihydrotanshinone I (DHTS) on human gastric cancer cells. The results showed that DHTS significantly inhibited the viability of AGS gastric cancer cells in a dose- and time-dependent manner by inducing reactive oxygen species generation, oxidative stress, and apoptosis. DHTS treatment led to elevated intracellular ROS, decreased glutathione levels, increased apoptotic cells, and activation of caspase-3 and caspase-8. Blocking ROS generation reversed DHTS-induced apoptosis. Therefore, DHTS exhibits anticancer effects in gastric cancer by initiating ROS-mediated oxidative stress and apoptosis.
The document describes screening methods for new anticancer drugs. It discusses how cancer arises from genetic mutations and different cancer types. Current treatments include chemotherapy, surgery and radiation. There is a need for more selective anticancer agents due to drug resistance and side effects. Various in vitro and in vivo screening assays are described to test compounds for cytotoxicity against cancer cells and tumors in animal models. The goal is to develop more effective and safer anticancer drugs.
This document summarizes a study that synthesized lipid carriers containing the anticancer drug doxorubicin (Dox) and tested their efficacy on HeLa and HCT116 cells. The carriers were around 200 nm in size. Treatment with 1 μM Dox delivered via the new carriers decreased survival of resistant HeLa cells by over 50%, while the blank carriers did not impair survival of sensitive HCT116 cells. This validated assay shows potential for determining efficacy of drug delivery systems.
The document summarizes various methods used to measure apoptosis and cell viability, including membrane integrity assays, functional assays, DNA labeling assays, and morphological assays. Membrane integrity assays like trypan blue exclusion, annexin V binding, and LDH leakage determine cell viability based on plasma membrane integrity. Functional assays like MTT, XTT, and Alamar Blue measure cell metabolism. DNA labeling assays use fluorescent conjugates to label cells, while morphological assays examine changes in cell morphology during apoptosis. The document provides details on the principles, materials, and procedures for several common assays used to measure apoptosis and cell viability.
DOI:10.21276/ijlssr.2016.2.4.2
neoplastic progression through the action of viral oncoproteins, mainly E6 and E7.Cervical cancer remains the second
most common cancer in women worldwide with India as a major contributor to global burden with an annual incidence of
132,000 new cases and mortality rate of 74,000 deaths annually. In this study turmeric, neem, tulasi and ginger were
selected as natural anticancer drugs. The objective of the study was to analyze the anticancer property of turmeric
(Curcuma longa), neem (Azadirachta indica), tulasi (Occimum sanctum) and ginger (Zingiber officinale) on HeLa cells.
Turmeric, neem, tulasi and ginger capsules (Himalaya’s Company) were used and aqueous and methanolic extracts of the
turmeric, neem, tulasi and ginger were obtained using a soxhlet extraction. To check the efficacy of these drug MTT assay
was performed, that determines % viability and/or cytotoxicity. IC50 of aqueous turmeric, neem, tulasi and ginger extracts
in case of HeLa cells were 17.8, 22, 79.4, 27.86 respectively and in case of methanolic turmeric, neem, tulasi and ginger
extracts 17, 7.35, 75.24 and 16.1 respectively. To confirm apoptosis as the sole reason behind cell death
immunofluorescence based apoptosis assay was performed using TALI image based cytometer. The study has led to
postulate hypothesis that natural drugs e.g. turmeric, neem, tulasi and ginger are potent anti-cancer compound that are
capable of inhibiting the growth of immortal cells by apoptosis. Key-words- Cervical cancer, Human papillomavirus (HPV), Oncoproteins E6 and E7, Natural compounds, HeLa cell
line (adherent), Cell viability and MTT assay, Apoptosis assay
1) The study investigated the effects of the tyrosine kinase inhibitors crizotinib and dasatinib on canine osteosarcoma cell lines. It found that dasatinib more effectively suppressed cell viability and inhibited hepatocyte growth factor-induced invasion and migration compared to crizotinib.
2) Exogenous hepatocyte growth factor increased invasion, migration, and matrix metalloproteinase production in the osteosarcoma cell lines.
3) Preliminary results showed that four dogs with osteosarcoma treated with adjuvant dasatinib therapy experienced an apparent clinical benefit.
This document discusses several in vitro methods for assessing the cytotoxicity of chemotherapeutic drugs, including assays using brine shrimp, MTT, sulforhodamine B, trypan blue dye, and acridine orange/ethidium bromide staining. It describes maintaining cell lines from cervical carcinoma and breast adenocarcinoma in culture, assessing cell viability, and preserving cells in liquid nitrogen. Specific methods are provided for assays including brine shrimp lethality, MTT, sulforhodamine B, trypan blue dye exclusion, acridine orange/ethidium bromide staining, and DNA fragmentation to evaluate the cytotoxic effects of test compounds on cultured malignant cell lines.
This document describes a screen of 50,000 compounds to identify inhibitors of hepatocyte growth factor (HGF)-induced epithelial scattering. Several structurally distinct compounds were identified that had not previously been reported to have biological activity. Further analysis revealed that many of the compounds broadly inhibit cancer cell proliferation by arresting cell division in G2/M phase with minimal induction of apoptosis. This effect is consistent with microtubule-targeting agents. Biochemical assays showed that several compounds directly inhibit microtubule polymerization. Some pharmacokinetic properties of the compounds were also assessed.
This study investigated the effects of dihydrotanshinone I (DHTS) on human gastric cancer cells. The results showed that DHTS significantly inhibited the viability of AGS gastric cancer cells in a dose- and time-dependent manner by inducing reactive oxygen species generation, oxidative stress, and apoptosis. DHTS treatment led to elevated intracellular ROS, decreased glutathione levels, increased apoptotic cells, and activation of caspase-3 and caspase-8. Blocking ROS generation reversed DHTS-induced apoptosis. Therefore, DHTS exhibits anticancer effects in gastric cancer by initiating ROS-mediated oxidative stress and apoptosis.
The document describes screening methods for new anticancer drugs. It discusses how cancer arises from genetic mutations and different cancer types. Current treatments include chemotherapy, surgery and radiation. There is a need for more selective anticancer agents due to drug resistance and side effects. Various in vitro and in vivo screening assays are described to test compounds for cytotoxicity against cancer cells and tumors in animal models. The goal is to develop more effective and safer anticancer drugs.
This document summarizes a study that synthesized lipid carriers containing the anticancer drug doxorubicin (Dox) and tested their efficacy on HeLa and HCT116 cells. The carriers were around 200 nm in size. Treatment with 1 μM Dox delivered via the new carriers decreased survival of resistant HeLa cells by over 50%, while the blank carriers did not impair survival of sensitive HCT116 cells. This validated assay shows potential for determining efficacy of drug delivery systems.
The document summarizes various methods used to measure apoptosis and cell viability, including membrane integrity assays, functional assays, DNA labeling assays, and morphological assays. Membrane integrity assays like trypan blue exclusion, annexin V binding, and LDH leakage determine cell viability based on plasma membrane integrity. Functional assays like MTT, XTT, and Alamar Blue measure cell metabolism. DNA labeling assays use fluorescent conjugates to label cells, while morphological assays examine changes in cell morphology during apoptosis. The document provides details on the principles, materials, and procedures for several common assays used to measure apoptosis and cell viability.
In vitro methods of screening of anticancer agentsNikitaSavita
Cancer- It is the leading cause of mortality. Cancer is the disease which is categorized by abnormal cell growth with the dormant to spread to other parts of body.
This document describes a cross-species gene expression methodology for comparing drug responses between species. Key points:
1) The methodology identifies orthologous gene probes between rat and human microarrays to develop a "virtual human" array from rat gene expression data. This allows direct comparison of rat and human drug responses at a molecular level.
2) Application of the method to a PPARα agonist showed conserved induction of marker genes between rat and virtual human arrays, though human response is typically weaker.
3) Analysis using artificial neural networks identified both shared and distinct gene expression markers of drug response in rat versus virtual human data, demonstrating the ability to find "bridge markers" for cross-species extrapolation.
This document discusses various methods for testing the mutagenicity of chemicals, including both prokaryotic and eukaryotic cell systems. It describes the Ames test which uses Salmonella bacteria to identify mutagens, as well as other prokaryotic methods like the host-mediated assay and coliform assay. Eukaryotic methods discussed include the Saccharomyces forward mutation assay, mammalian cell tests, and in vivo assays like the micronucleus test and dominant lethal assay. The document provides details on the procedures and principles of many of these important mutagenicity testing methods.
This document discusses the preclinical evaluation of anticancer agents. In vitro cytotoxicity studies are conducted first using various assays like MTT, SRB, dye exclusion tests, etc. on cancer cell lines. Promising compounds are then evaluated in in vivo tumor models using mice. Acute toxicity studies in mice and dogs help determine the safe starting dose for clinical trials. Various tumor models used include chemically induced cancers like DMBA-induced mouse skin papillomas and MNU-induced rat mammary gland cancer, as well as transplantable murine and human tumor models. Together, these preclinical studies aim to identify candidate anticancer agents and guide their safe entry into clinical trials.
This document discusses a study that found significant differences in gene expression variability between knockout and wild-type mice using microarray data from 25 publicly available datasets. The study found that knockouts exhibited either significantly increased or decreased variability compared to wild-types in virtually every dataset analyzed. Examination of the data distributions indicated that these differences were due to broad changes in variability across most genes, rather than being driven by outliers. The findings suggest that changes in gene expression variability due to gene knockouts may have important phenotypic consequences.
This document discusses screening methods for anticancer agents. It describes both in vitro and in vivo methods. For in vitro screening, assays are discussed that measure cell viability, proliferation, and cytotoxicity, such as the MTT assay, Sulforhodamine B assay, 3H-thymidine uptake assay, and dye exclusion tests. For in vivo screening, models are described that use chemically-induced tumors in animals as well as cell line and xenograft transplant models to test potential anticancer agents and measure effects on tumor growth.
This study evaluated the in vitro skin phototoxicity of cosmetic formulations containing photounstable and photostable UV filters (octyl methoxycinnamate, benzophenone-3, avobenzone, octyl salicylate, 4-methylbenzilidene camphor) and vitamin A palmitate using two tests: the 3T3 Neutral Red Uptake Phototoxicity Test and the Human 3-D Skin Model In Vitro Phototoxicity Test. Avobenzone showed pronounced phototoxicity and vitamin A showed a tendency toward weak phototoxic potential. A synergistic effect of vitamin A palmitate on the phototoxicity of combinations containing avobenzone was observed
Echinocandin susceptibility testing of candida species comparison of eucast ...Abdul Rahim Akbar
This study compared nine antifungal susceptibility testing methods for their ability to distinguish between Candida isolates with known mutations conferring echinocandin resistance (FKS hot spot mutants, n=29) and wild-type isolates (n=94). The methods included EUCAST, CLSI microdilution, agar dilution, Etest, and disk diffusion using RPMI and IsoSensitest media. For the three echinocandins tested (anidulafungin, caspofungin, micafungin), the reference methods EUCAST and CLSI, as well as agar dilution and Etest with RPMI medium, best separated the mutant and wild-type populations with few errors. However,
V79C cells were derived from V79 cell line by through chronic oxidative stress that was found to be radio-resistant. These cells had demonstrated transformation–like stable changes and could be used as model system to study oxidant induced carcinogenesis. Our objective was to understand mechanism of radiation-resistance in these cells. Apoptotic cell death was inhibited in these cells as visualized microscopically by Hoechst staining and nucleosomal ladder formation in agarose gel. Release of cytochrome c in cytoplasm and Apoptosis Inducing Factor in the mitochondrial and nuclear fraction of cells were determined by Western blotting. The caspase 9 and caspase 3 activities in these cells were estimated from fluorimetric assays. These results revealed that the radiation resistance was due to inhibition of caspase-dependent apoptotic death pathways although Apoptosis Inducing Factor mediated pathway remained unaffected. These findings may aid in understanding the mechanism of radiation resistance in tumors arising from oxidative stress.
In-vitro evaluation techniques of anticancer, anti oxidant, anti microbial ZakiyaUsmani
This document discusses various in vitro methods used to evaluate potential anti-cancer and antioxidant compounds, as well as antimicrobial activity. It describes cytotoxicity assays such as MTT, SRB, clonogenic assays and dye exclusion tests that are used to study anti-cancer activity against cell lines. Methods to evaluate antioxidant activity in vitro include DPPH radical scavenging, hydrogen peroxide and superoxide radical scavenging assays. Diffusion and dilution methods are discussed for determining antimicrobial activity of compounds in vitro prior to animal studies.
The document discusses various methods for evaluating anticancer activity and cell viability, including membrane integrity assays, functional assays, and DNA assays. Membrane integrity assays like trypan blue exclusion and LDH leakage are commonly used to determine cell viability by measuring intact cell membranes. Additional assays measure cellular functions, DNA integrity, morphology, and reproductive ability.
This is a lecture by Dr. Jerry McLaughlin about his research into extracts of pawpaw plants, annonaceous acetogenins, in vitro, in vivo, mechanism of action, and toxicity in mice.
Cancer is characterized by uncontrolled cell proliferation that has transformed from normal cells. Several screening methods are used to test potential cancer treatments in vitro and in vivo. In vitro methods include tetrazolium salt assays, sulphorhodamine B assays, and thymidine uptake assays to test cell viability. In vivo methods include inducing tumors in mice and rats through chemicals like DMBA and testing whether treatments reduce tumor incidence and size. DMBA is used to induce skin papillomas in mice and mammary gland carcinomas in rats, with the drug's efficacy measured by decreased tumor rates compared to controls. Various assays then measure cell viability and proliferation after treatment to screen for potential anti-cancer effects.
Anti- Tumor assay / Screening of Anticancer DrugsPratik Parikh
This document presents information about in vitro and in vivo anti-tumor assays. It discusses several in vitro assays including tetrazolium salt, sulphorhodamine B, and 3H-thymidine uptake assays. It also describes various in vivo models like carcinogen-induced tumors in mice, viral infection models, transplantation models, and genetically engineered mouse models. Specific techniques covered include DMBA-induced skin papillomas in mice and MNU-induced rat mammary gland cancer models. The document concludes by discussing parameters to evaluate anti-tumor effects in an EAC liquid tumor mouse model.
This is Part 1 of a presentation on Genetic Toxicology that was given by Dr. David Kirkland to scientific staff at Health Canada in Sept. 2010. Part 2 is availabile in ppt
This document summarizes a proteomic analysis of the excretory/secretory (ES) proteins of Toxoplasma gondii, the causative agent of toxoplasmosis. 34 proteins were identified from the ES proteins of the RH strain of T. gondii using LC/MS/MS analysis and spectral counting methods. The three most abundant proteins identified were GRA1, GRA7, and GRA2. A variety of other proteins were also identified, including micronemal proteins, rhoptry proteins, and dense granule proteins, which play important roles in T. gondii host cell invasion and survival within host cells. This analysis provides new insights into the ES proteome of T
IOSRPHR(www.iosrphr.org) IOSR Journal of Pharmacyiosrphr_editor
This study investigated extended spectrum beta-lactamase (ESBL) producing Escherichia coli at a rural medical college in Himachal Pradesh, India. The study found a high frequency of ESBL production in E. coli clinical isolates. Of the 175 E. coli isolates tested, 45% were confirmed to be ESBL producers using double disc synergy testing, while 23% were confirmed using disc on disc testing. The isolates showed high resistance to many first-line antibiotics but were uniformly susceptible to meropenem. The results demonstrate the need for prudent antibiotic use and mandatory ESBL detection testing to inform treatment and control spread.
The Radiosensitivity Effect of Hydroxyurea on HT29 Cell Lineiosrphr_editor
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
This document discusses screening methods for anti-cancer agents. It begins with an introduction to cancer, noting that cancer is caused by uncontrolled cell proliferation due to genetic mutations from carcinogens. It then discusses the need for novel anti-cancer drugs due to issues like drug resistance and side effects. The document outlines several in vitro and in vivo screening methods, including membrane integrity assays, functional assays, DNA labeling assays, morphological assays, and reproductive assays. Specific assays discussed in more detail include MTT, LDH, annexin V/PI, trypan blue, and colony forming assays. The document provides details on the principles, advantages and disadvantages of these various screening methods.
Cardiovascular disease is the leading cause of early death among diabetes with 70 per cent dying of blood coagulation and 65 per cent from heart disease and stroke. A balanced diet coupled with physical activity and a healthy lifestyle will keep cardiovascular diseases at bay. To know about the risk factors and measures one can take to counter them, read on…
In vitro methods of screening of anticancer agentsNikitaSavita
Cancer- It is the leading cause of mortality. Cancer is the disease which is categorized by abnormal cell growth with the dormant to spread to other parts of body.
This document describes a cross-species gene expression methodology for comparing drug responses between species. Key points:
1) The methodology identifies orthologous gene probes between rat and human microarrays to develop a "virtual human" array from rat gene expression data. This allows direct comparison of rat and human drug responses at a molecular level.
2) Application of the method to a PPARα agonist showed conserved induction of marker genes between rat and virtual human arrays, though human response is typically weaker.
3) Analysis using artificial neural networks identified both shared and distinct gene expression markers of drug response in rat versus virtual human data, demonstrating the ability to find "bridge markers" for cross-species extrapolation.
This document discusses various methods for testing the mutagenicity of chemicals, including both prokaryotic and eukaryotic cell systems. It describes the Ames test which uses Salmonella bacteria to identify mutagens, as well as other prokaryotic methods like the host-mediated assay and coliform assay. Eukaryotic methods discussed include the Saccharomyces forward mutation assay, mammalian cell tests, and in vivo assays like the micronucleus test and dominant lethal assay. The document provides details on the procedures and principles of many of these important mutagenicity testing methods.
This document discusses the preclinical evaluation of anticancer agents. In vitro cytotoxicity studies are conducted first using various assays like MTT, SRB, dye exclusion tests, etc. on cancer cell lines. Promising compounds are then evaluated in in vivo tumor models using mice. Acute toxicity studies in mice and dogs help determine the safe starting dose for clinical trials. Various tumor models used include chemically induced cancers like DMBA-induced mouse skin papillomas and MNU-induced rat mammary gland cancer, as well as transplantable murine and human tumor models. Together, these preclinical studies aim to identify candidate anticancer agents and guide their safe entry into clinical trials.
This document discusses a study that found significant differences in gene expression variability between knockout and wild-type mice using microarray data from 25 publicly available datasets. The study found that knockouts exhibited either significantly increased or decreased variability compared to wild-types in virtually every dataset analyzed. Examination of the data distributions indicated that these differences were due to broad changes in variability across most genes, rather than being driven by outliers. The findings suggest that changes in gene expression variability due to gene knockouts may have important phenotypic consequences.
This document discusses screening methods for anticancer agents. It describes both in vitro and in vivo methods. For in vitro screening, assays are discussed that measure cell viability, proliferation, and cytotoxicity, such as the MTT assay, Sulforhodamine B assay, 3H-thymidine uptake assay, and dye exclusion tests. For in vivo screening, models are described that use chemically-induced tumors in animals as well as cell line and xenograft transplant models to test potential anticancer agents and measure effects on tumor growth.
This study evaluated the in vitro skin phototoxicity of cosmetic formulations containing photounstable and photostable UV filters (octyl methoxycinnamate, benzophenone-3, avobenzone, octyl salicylate, 4-methylbenzilidene camphor) and vitamin A palmitate using two tests: the 3T3 Neutral Red Uptake Phototoxicity Test and the Human 3-D Skin Model In Vitro Phototoxicity Test. Avobenzone showed pronounced phototoxicity and vitamin A showed a tendency toward weak phototoxic potential. A synergistic effect of vitamin A palmitate on the phototoxicity of combinations containing avobenzone was observed
Echinocandin susceptibility testing of candida species comparison of eucast ...Abdul Rahim Akbar
This study compared nine antifungal susceptibility testing methods for their ability to distinguish between Candida isolates with known mutations conferring echinocandin resistance (FKS hot spot mutants, n=29) and wild-type isolates (n=94). The methods included EUCAST, CLSI microdilution, agar dilution, Etest, and disk diffusion using RPMI and IsoSensitest media. For the three echinocandins tested (anidulafungin, caspofungin, micafungin), the reference methods EUCAST and CLSI, as well as agar dilution and Etest with RPMI medium, best separated the mutant and wild-type populations with few errors. However,
V79C cells were derived from V79 cell line by through chronic oxidative stress that was found to be radio-resistant. These cells had demonstrated transformation–like stable changes and could be used as model system to study oxidant induced carcinogenesis. Our objective was to understand mechanism of radiation-resistance in these cells. Apoptotic cell death was inhibited in these cells as visualized microscopically by Hoechst staining and nucleosomal ladder formation in agarose gel. Release of cytochrome c in cytoplasm and Apoptosis Inducing Factor in the mitochondrial and nuclear fraction of cells were determined by Western blotting. The caspase 9 and caspase 3 activities in these cells were estimated from fluorimetric assays. These results revealed that the radiation resistance was due to inhibition of caspase-dependent apoptotic death pathways although Apoptosis Inducing Factor mediated pathway remained unaffected. These findings may aid in understanding the mechanism of radiation resistance in tumors arising from oxidative stress.
In-vitro evaluation techniques of anticancer, anti oxidant, anti microbial ZakiyaUsmani
This document discusses various in vitro methods used to evaluate potential anti-cancer and antioxidant compounds, as well as antimicrobial activity. It describes cytotoxicity assays such as MTT, SRB, clonogenic assays and dye exclusion tests that are used to study anti-cancer activity against cell lines. Methods to evaluate antioxidant activity in vitro include DPPH radical scavenging, hydrogen peroxide and superoxide radical scavenging assays. Diffusion and dilution methods are discussed for determining antimicrobial activity of compounds in vitro prior to animal studies.
The document discusses various methods for evaluating anticancer activity and cell viability, including membrane integrity assays, functional assays, and DNA assays. Membrane integrity assays like trypan blue exclusion and LDH leakage are commonly used to determine cell viability by measuring intact cell membranes. Additional assays measure cellular functions, DNA integrity, morphology, and reproductive ability.
This is a lecture by Dr. Jerry McLaughlin about his research into extracts of pawpaw plants, annonaceous acetogenins, in vitro, in vivo, mechanism of action, and toxicity in mice.
Cancer is characterized by uncontrolled cell proliferation that has transformed from normal cells. Several screening methods are used to test potential cancer treatments in vitro and in vivo. In vitro methods include tetrazolium salt assays, sulphorhodamine B assays, and thymidine uptake assays to test cell viability. In vivo methods include inducing tumors in mice and rats through chemicals like DMBA and testing whether treatments reduce tumor incidence and size. DMBA is used to induce skin papillomas in mice and mammary gland carcinomas in rats, with the drug's efficacy measured by decreased tumor rates compared to controls. Various assays then measure cell viability and proliferation after treatment to screen for potential anti-cancer effects.
Anti- Tumor assay / Screening of Anticancer DrugsPratik Parikh
This document presents information about in vitro and in vivo anti-tumor assays. It discusses several in vitro assays including tetrazolium salt, sulphorhodamine B, and 3H-thymidine uptake assays. It also describes various in vivo models like carcinogen-induced tumors in mice, viral infection models, transplantation models, and genetically engineered mouse models. Specific techniques covered include DMBA-induced skin papillomas in mice and MNU-induced rat mammary gland cancer models. The document concludes by discussing parameters to evaluate anti-tumor effects in an EAC liquid tumor mouse model.
This is Part 1 of a presentation on Genetic Toxicology that was given by Dr. David Kirkland to scientific staff at Health Canada in Sept. 2010. Part 2 is availabile in ppt
This document summarizes a proteomic analysis of the excretory/secretory (ES) proteins of Toxoplasma gondii, the causative agent of toxoplasmosis. 34 proteins were identified from the ES proteins of the RH strain of T. gondii using LC/MS/MS analysis and spectral counting methods. The three most abundant proteins identified were GRA1, GRA7, and GRA2. A variety of other proteins were also identified, including micronemal proteins, rhoptry proteins, and dense granule proteins, which play important roles in T. gondii host cell invasion and survival within host cells. This analysis provides new insights into the ES proteome of T
IOSRPHR(www.iosrphr.org) IOSR Journal of Pharmacyiosrphr_editor
This study investigated extended spectrum beta-lactamase (ESBL) producing Escherichia coli at a rural medical college in Himachal Pradesh, India. The study found a high frequency of ESBL production in E. coli clinical isolates. Of the 175 E. coli isolates tested, 45% were confirmed to be ESBL producers using double disc synergy testing, while 23% were confirmed using disc on disc testing. The isolates showed high resistance to many first-line antibiotics but were uniformly susceptible to meropenem. The results demonstrate the need for prudent antibiotic use and mandatory ESBL detection testing to inform treatment and control spread.
The Radiosensitivity Effect of Hydroxyurea on HT29 Cell Lineiosrphr_editor
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
This document discusses screening methods for anti-cancer agents. It begins with an introduction to cancer, noting that cancer is caused by uncontrolled cell proliferation due to genetic mutations from carcinogens. It then discusses the need for novel anti-cancer drugs due to issues like drug resistance and side effects. The document outlines several in vitro and in vivo screening methods, including membrane integrity assays, functional assays, DNA labeling assays, morphological assays, and reproductive assays. Specific assays discussed in more detail include MTT, LDH, annexin V/PI, trypan blue, and colony forming assays. The document provides details on the principles, advantages and disadvantages of these various screening methods.
Cardiovascular disease is the leading cause of early death among diabetes with 70 per cent dying of blood coagulation and 65 per cent from heart disease and stroke. A balanced diet coupled with physical activity and a healthy lifestyle will keep cardiovascular diseases at bay. To know about the risk factors and measures one can take to counter them, read on…
The abdominal cocoon syndrome is described as a rare entity in which part or whole of the small bowel is enclosed in a fibrous membrane. This case report is of a 35 years old woman who had a provisional diagnosis of ovarian cyst. Intraoperatively she was found to have abdominal cocoon syndrome. Laparotomy with cystectomy was done. She developed subacute intestinal obstruction 5 days later. This was managed conservatively.
Type 2 Diabetes Mellitus: The Concerned Complications and Target OrgansApollo Hospitals
Diabetes has been considered as the most dreaded non-communicable disease consuming the mankind rapidly. WHO has predicted the number of diabetics to be approximately 366 millions by 2030. The disease is characterized by hyperglycemia and the basic symptoms are polyphagia, polydipsia and polyuria. The autoimmune type 1 diabetes represent almost 1% of the total diabetic population, the rest being that of type 2 diabetes (T2D). Type 2 diabetes has been linked to a variety of factors such as heredity, environmental factors, unhealthy eating habits, sedentary lifestyle, stress etc. The uncontrolled hyperglycemia has profound deleterious effects on almost all the organs and results in various cardiovascular disorders, retinopathy, neuropathy, and nephropathy. Recent studies have revealed an array of pulmonary dysfunctions related with T2D ranging from respiratory defects to tuberculosis. Diabetes also predisposes the person to hepatic dysfunctions like NAFLD and HCC and a range of infections at various sites which are difficult to manage. Post-surgical infections are of special interest for subjects with uncontrolled hyperglycemia prior to surgery. Scientists all over the world are revealing different pathways and associated therapies for type 2 diabetes in order to control the pathological effects covering almost whole body physiology.
Therapeutic plasma exchange in patients with neurologic diseases: Retrospecti...Apollo Hospitals
Therapeutic plasma exchange (TPE) is commonly used in many neurological disorders where an immune aetiology was known or suspected. We report our experience with TPE performed for neuroimmunologic disorders at four university hospitals.
There are 150,000 to 200,000 children born each year with congenital heart disease in India. Apollo Clinics can now, using advanced technology find out whether or not a baby has a heart problem, even when the mother is pregnant. Tests can be performed at 18 weeks of gestation to decide on a course of action.
In a pregnant woman presenting with thrombocytopenia, the possibility of HELLP syndrome should always be considered by the treating clinician so as to initiate the therapy at the earliest to prevent the high perinatal mortality and postpartum morbidity. Here we report an unusual case of young Primigravida (postpartum) who presented at Indraprastha Apollo hospitals, New Delhi with altered sensorium, paraperesis, DIC and septic shock. On evaluation she was found to have HELLP syndrome for which Plasmapheresis was given and patient showed remarkable improvement.
Burden of cardiovascular diseases in Indians: Estimating trends of coronary a...Apollo Hospitals
The global trends in disease specific mortalities indicate that ischemic heart disease (IHD) is the leading cause of death in age group ≥60 years. It is also being recognized that cardiovascular diseases (CVDs) and their risk factors are emerging as primary health problems in India with all socioeconomic groups being equally vulnerable. Though the high mortality rates due to CVDs in India may have major economic repercussions, the analysis on economic impact of CVDs remains incomplete, because of inadequate coverage of these diseases in India's vital event registration and absence of surveillance systems for disease specific mortality data. The per capita expenditure on health by public sector is very low making the poor to go for costly private healthcare facilities. We discuss here the burden of CAD and its risk factors in India and need for using population and individual based prevention strategies to halt and reverse the CVD epidemic. The country will need to create data for technical and operational factors for making prevention and control of CVDs feasible. National and international multidisciplinary collaborations will be needed to address the challenge posed by CVDs.
High blood pressure or BP or hypertension has been termed as the ‘silent killer’. It is essential to know in detail about this silent killer and take measures not to fall prey to it. The pressure in your blood varies depending on the type of work you are doing.
The document provides three recipes for reducing weight that use low glycemic index carbohydrates: moong dal tikki, sizzling mushrooms in salsa, and cottage cheese and spinach balls. The recipes call for ingredients like yellow moong dal, mushrooms, cottage cheese, and spinach that when combined, cooked, and eaten can help with weight loss and maintaining long term health by producing only small fluctuations in blood glucose and insulin levels. Apollo Clinics dieticians can suggest more customized recipes and diet charts to aid in weight loss goals.
Maternal floor infarction: A rare cause of sudden intrauterine fetal demiseApollo Hospitals
Maternal floor infarction is a rare placental lesion in which large amounts of fibrin are deposited along the basal plate, which becomes avascular and sclerotic. The rate of fetal salvage is very poor as the lesion develops rapidly.
Parasitic infection and immunomodulation: A possible explanation for the hygi...Apollo Hospitals
This document discusses the hygiene hypothesis in autoimmune and allergic disease. It proposes that reduced incidence of parasitic infections in developed countries due to improved sanitation may be linked to increased rates of autoimmune and allergic diseases. Parasitic infections induce regulatory immune responses that help the parasites survive while also reducing inflammation. Specific parasite molecules modulate the immune system by suppressing Th1 and Th17 responses and inducing Th2 and regulatory T cell responses. Understanding these immunomodulatory mechanisms could help develop new treatments for inflammatory and allergic conditions.
This document reports on three siblings diagnosed with Wolfram Syndrome, also known as DIDMOAD syndrome, which is a rare genetic disorder characterized by diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. The siblings presented with these symptoms at different ages. Clinical findings and investigations confirmed the diagnosis in all three cases. The document discusses the pathogenesis and clinical manifestations of Wolfram Syndrome.
This study analyzed loss of heterozygosity (LOH) in papillary thyroid carcinoma (PTC) by examining microsatellite markers in the M6P/IGFIIR gene region and three loci on chromosome 11 in 98 PTC tumor and normal tissue samples. The samples showed 49-63% heterozygosity for the markers analyzed. However, LOH was not detected in any of the informative heterozygous samples, indicating that LOH did not contribute to PTC development in these patients for the regions studied.
This study investigated the effects of high-dose ascorbic acid (ASC) alone and in combination with the drug sorafenib (SF) on liver (HepG2), brain (SKNSH), and kidney (HEK) cell lines. ASC at 5mM caused more cell death than other compounds in SKNSH and HepG2 cells. ASC combined with lower doses of SF produced greater cytotoxic effects than other combinations in SKNSH cells. While ASC and SF individually killed cancer cells in a dose-dependent manner, very high doses of ASC up to 100mM did not kill non-cancerous HEK kidney cells. The results suggest that high-dose ASC alone or with lower doses of SF
Principles of cell viability assays by surendra.pptxSurendra Chowdary
1.DYE EXCLUSION ASSAYS:
Dye exclusion assays are the simplest methods that are based on utilization of different dyes such as trypan blue, eosin, congo red, and erythrosine B, which are excluded by the living cells, but not by dead cells.
For these assays, although staining procedure is quite straightforward, experimental procedure may be time-consuming in case of large sample sizes.
a. Trypan blue stain assay:
Trypan blue stain assay has initially been developed in 1975 to measure viable cell count and is still used as a confirmatory test for measuring changes in viable cell number caused by a drug or toxin.
Trypan blue stain, a large negatively charged molecule, is one of the simplest assays that are used to determine the number of viable cells in a cell suspension.
Principle:
The principle of this assay is that living cells have intact cell membranes that exclude the trypan blue stain, whereas dead cells do not.
Cell suspension is mixed with the trypan blue stain and examined visually under light microscopy to determine whether cells include or exclude the stain.
A viable cell will have a clear cytoplasm, whereas a nonviable cell will have a blue cytoplasm.
Reagent preparation:
To perform the trypan blue stain assay, 0.4% trypan blue stain and phosphate- buffered saline (PBS) or serum-free medium are obtained.
Trypan blue stain should be stored in dark and filtered after prolonged storage.
As trypan blue stain binds to serum proteins and causing misleading results, serum-free medium should be used to obtain reliable results.
Assay Protocol:
The cell suspension to be tested is centrifuged at 100 g for 5 min.
The supernatant is discarded and the pellet is resuspended in 1-ml PBS solution or serum-free medium.
Then, one portion of this cell suspension is mixed with one portion of trypan blue stain.
The mixture is allowed to stay at room temperature for 3 min. It is important to note that the cells should be counted within 3–5 min of mixing with trypan blue, as longer incubation periods will lead to cell death and hence reduced viability counts.
Following the incubation, a drop of the mixture is applied to a hemocytometer, which is placed on the stage of a binocular microscope.
Viable cells will remain unstained, and nonviable cells will stain, in the hemocytometer and these cells are counted separately.
.
Calculation:
After counting viable and nonviable cells, the total number of viable cells per milliliter of aliquot is determined by multiplying the total number of viable cells by 2, which is the dilution factor for trypan blue.
Similarly, total number of cells per milliliter of aliquot is determined by addition of number of viable and nonviable cells and multiplying it by 2.
Then, the percentage of viable cells is calculated using the following equation.
% Viable cells = Total number of viable cells per milliliter of aliquot × 100.
Total number of cells per milliliter of aliquot
2.COLORIMETRIC ASSAYS:
Colorimetric assays
The document summarizes research finding that anaplastic large cell lymphoma contains a small subset of cells, detectable by side population analysis, that have tumor-propagating properties. Specifically:
1) A small proportion (1-3.5%) of cells from ALCL cell lines and patient tumors were found to efflux dye and form a side population, enriched for the ABCG2 transporter.
2) These side population cells were shown to proliferate in vitro and in vivo, regenerating both themselves and the bulk tumor population.
3) Limiting dilution transplantation experiments in mice determined the frequency of tumor-propagating cells to be as high as 1 in 54 cells for cell lines and 1 in 1
Principles & Applications of cell viability assays (MTT Assays)VidyaNani
This document discusses cell viability assays, specifically focusing on principles and applications of MTT assays. It defines cell viability and describes various types of cell viability assays including dye exclusion, colorimetric, fluorometric, luminometric, and flow cytometric assays. The document provides details on the MTT assay, including its principle, protocol, and applications for measuring cell proliferation, cytotoxicity, and metabolic activity. The MTT assay is a common colorimetric assay that measures the reduction of yellow MTT by mitochondrial dehydrogenases in viable cells to produce purple formazan crystals.
This document summarizes research presented on isolating and characterizing human fetal liver stem cells. The key steps involved isolating stem cells from human fetal liver tissue using markers like AFP, CK18, and albumin. The isolated stem cells were then characterized using techniques like cell counting, viability assays, immunocytochemistry, MTT assays, and PCR to analyze RNA expression and confirm the presence of stem cell markers. The overall aim was to isolate fetal liver stem cells and characterize their properties for potential applications in areas like blood transfusions, organ transplantation, and disease research.
Clinical diagnosis of chronic myeloid leukemia by real time polymerase chain ...Teboho Mooko
Oncology study i did in my third year (2014). the study was basically about monitoring Chronic Myeloid leukemia (CML) using Real-Time PCR techniques to check how patients from Universitas Hospital responded to the treatment of Gleevec drug.
This document summarizes an in-vitro study that assessed the cytotoxicity of halloysite nanotubes against two cancer cell lines (HepG2 and HCT116) and human peripheral blood lymphocytes. The halloysite nanotubes were first characterized using transmission electron microscopy, which found that over 50% were 500 nm in length, 90% were less than 150 nm in diameter, and over 90% had an aspect ratio of less than 12. In vitro assays then evaluated the cytotoxicity of the halloysite nanotubes against the two cancer cell lines and human lymphocytes at concentrations up to 1000 μg/mL over 24, 48 and 72 hours. The results collectively indicated that the halloysite nanotubes were
The document summarizes research finding that anaplastic large cell lymphoma (ALCL) cell lines and primary patient tumors contain a small subset of cells, termed side population (SP) cells, that have tumor-propagating capacity. Specifically:
1) SP cells ranging from 1-3.5% were identified in ALCL cell lines and 0.4-3% in primary tumors using dye efflux and marker expression.
2) Isolated SP cells were shown to proliferate in vitro, regenerate the bulk tumor population over time, and support growth of non-SP cells through soluble factors.
3) In mouse models, as few as 50 SP cells were able to form tumors, whereas thousands
This document describes research investigating the potential of human endometrial stem cells (EnSCs) to differentiate into neural cells. EnSCs were isolated from endometrial biopsies and characterized by flow cytometry, showing expression of stem cell markers and lack of hematopoietic/endothelial markers. The EnSCs were induced to differentiate into neural cells using growth factors like bFGF, PDGF, and EGF. After induction, the cells expressed neural markers like MAP2, β3-tubulin, and NF-L at the mRNA and protein levels based on RT-PCR and immunocytochemistry. The results demonstrate that EnSCs can respond to signaling molecules and differentiate into neuronal-like cells, suggesting their potential
Pluripotent Stem Cell Markers and microRNA Expression May Correlate with Dent...asclepiuspdfs
Background: Recent evidence has demonstrated that dental pulp stem cells (DPSC) may represent a source of pluripotent progenitors capable of differentiating into many cell and tissue types. Although microRNAs are known to modulate differentiation and function in human dental tissues, much of this research has focused selectively on tooth development. The primary objective of this study was to evaluate the expression of microRNA in dental pulp stem cell isolates to compare with classical biomarkers of cellular phenotypes and pluripotency. Materials and Methods: Using eight previously isolated and characterized DPSC isolates, growth and viability were evaluated and RNA was extracted for mRNA screening. DPSC biomarker and microRNA expression were analyzed for comparison with cellular phenotypes. Results: Evaluation of the growth and proliferation rates of each cell line resulted in the categorization of DPSC isolates into rapid, intermediate, and slow doubling times, which demonstrated higher viability among the most rapidly proliferating DPSCs. Analysis of DPSC biomarkers (Oct-4, Sox-2, NANOG) revealed an association with total live cell count, while microRNA expression (miR-27, miR-218, miR-124, and miR-16) appeared to be more closely associated with cellular viability. Conclusions: Although this study was limited to a small number of DPSC isolates, these results suggest a more thorough investigation and evaluation of biomarkers and microRNA expression may be necessary to elucidate the associations and complex interconnections with DPSC viability, proliferation, differentiation, and pluripotency.
This study investigated the binding targets of the inhaled anesthetic halothane in rat brain. The researchers used quantitative photoaffinity labeling and electrophoresis to analyze rat cerebellar homogenates exposed to [14C]halothane. They found that halothane incorporated into many soluble and membrane-bound proteins, with stoichiometry values ranging from 0 to 4 and apparent IC50 values from 0.2 to 2.0 mM. Competition experiments showed that unlabeled halothane, chloroform, and isoflurane inhibited halothane labeling to varying degrees, while a non-anesthetic compound inhibited the least. These results suggest that halothane binding motifs can be found in a wide variety of proteins in the brain
immunohistochemically and in situ hybridizati.pptxMarwa972783
This document discusses immunohistochemistry and in situ hybridization detection of COX2 in salivary gland tumors. It provides background on immunohistochemistry and in situ hybridization techniques. It then describes a study that examined COX2 expression in pleomorphic adenoma and mucoepidermoid carcinoma salivary gland tumor samples using immunohistochemistry. The study found higher COX2 expression in mucoepidermoid carcinoma samples compared to pleomorphic adenoma samples.
immunohistochemically and in situ hybridizati.pptxMarwa972783
This document discusses immunohistochemistry and in situ hybridization detection of COX2 in salivary gland tumors. It provides background on immunohistochemistry and in situ hybridization techniques. It then describes a study that examined COX2 expression in pleomorphic adenoma and mucoepidermoid carcinoma salivary gland tumor samples using immunohistochemistry. The study found higher COX2 expression in mucoepidermoid carcinoma samples compared to pleomorphic adenoma samples.
Evaluating the ability of anti-cancer drugs Etoposide and Staurosporine to in...Davient Bala
Cervical cancer is considered one of the most prevalent cancers affecting Singaporean women.
Although many novel chemotherapeutics have been developed recently, little has been done to
determine the efficiency of current anti-cancer agents working in combination. Here, we aimed to
evaluate the apoptosis induction efficiency of Etoposide and Staurosporine in HeLa cells. Cell cultures
were subjected to either 50 μM Etoposide, 10 nM Staurosporine or both for 24 hours prior visualization
under a fluorescence microscope. We found that Etoposide alone had an efficiency of 16.1% while
Staurosporine alone had 18.3%. However, the polytherapy achieved an efficiency of up to 33.6%,
which indicates an additive effect of both drugs to induce apoptosis. Our results demonstrate that
Etoposide and Staurosporine are both capable of inducing apoptosis in HeLa cells. Furthermore, it
reveals the potential of Etoposide-Staurosporine polytherapy to be a potent combinative treatment
option for cervical cancer patients resistant or sensitive to conventional anti-cancer agents.
This study utilized flow cytometric immunophenotyping to analyze bone marrow samples from 30 newly diagnosed myelodysplastic syndrome (MDS) patients. Abnormalities were frequently observed in the granulocytic, monocytic, and erythroid lineages of MDS patients compared to controls. The most common abnormality in granulocytes was decreased expression of CD10. Low expressions of CD13 were most common in monocytes. The erythroid lineage commonly showed low expression of CD71 and CD235a. Flow cytometry provided supportive evidence of phenotypic abnormalities to aid in the diagnosis of MDS when morphological analysis alone was inconclusive.
Publication - The Effect of Red-Allotrope Selenium Nanoparticles on Head and ...Christopher Hassan
This document summarizes a study that investigated the effect of red-allotrope selenium nanoparticles (rSeNPs) on head and neck squamous cell carcinoma (HNSCC) cells and human dermal fibroblast (HDF) cells. rSeNPs were synthesized and characterized, then HNSCC and HDF cells were exposed to various concentrations of rSeNPs for 1-3 days. Results showed that rSeNPs were approximately four times more cytotoxic to HNSCC cells than HDF cells. An effective dosage range for killing HNSCC cells while minimizing damage to HDF cells was established at 20-55 μg rSeNP/mL media over 3 days. Observations also showed low doses of rSe
Determination and comparison rate of expression markers of osteoblast derived...IJERD Editor
Nowadays high accident rates, fractures leading to permanent bone disorders and the impossibility of bone transplant have made scientists to look for new methods of repairing injured bones. Considering the application of stem cells in bone tissue engineering, there exists the necessity to investigate various culture methods and suitable fields and scaffolds. Thus, we decided to induce adipose-derived stem cells into osteoblast cells in two systems of pellet culture and monolayer and compare osteogenic markers. Methods: Stem cells have been separated via mechanical and enzymatic methods and cultured in monolayer and pellet culture models with osteogenic medium. Then, RNA was separated from differentiated cells, complementary DNA (cDNA) was synthesized and amplified. Polymerase chain reaction (PCR) product was transferred to electrophoresis gel. The intensity of the bands was measured by Image-J software and analyzed by SPSS.
This document discusses how normalization methods for gene expression measurements that assume equal cellular RNA content across experimental conditions can mask differences in total RNA yield per cell. The authors demonstrate that total RNA yield from Chinese Hamster Ovary (CHO) cells varies between experimental treatments and cell lines expressing recombinant proteins. They apply a normalization method using synthetic spike-in RNA standards added proportionally to cell number, which reveals differences in cellular RNA content and allows detection of global transcriptional amplification or repression. They use this method to assess gene expression in CHO cell lines of different sizes treated with cell cycle or mTOR inhibitors, or subjected to high osmolarity conditions. They find that a cell cycle inhibitor increases cell size and transcriptional amplification, an mTOR inhibitor causes
Similar to Development of cell culture samples for drug screening with bone marrow stem cells (20)
Movement disorders: A complication of chronic hyperglycemia? A case reportApollo Hospitals
A 77-year-old man presented with bilateral choreic movements that had developed over the past month. He had a history of poorly controlled type 2 diabetes. At admission, he was found to have severe hyperglycemia without ketosis. A CT scan showed hyperdensity in the putamen and lenticular nucleus. Treatment with insulin, haloperidol, and glycemic control led to regression of the choreic movements within 4 days. Chorea secondary to nonketotic hyperglycemia is a rare complication of uncontrolled diabetes that is usually reversible with normalization of blood glucose levels and neuroleptic treatment. The pathophysiology is thought to involve metabolic disturbances from hyperglycemia impairing neurotransmission in basal ganglia structures and
Malignant Mixed Mullerian Tumor – Case Reports and Review ArticleApollo Hospitals
Malignant mixed mullerian tumors are very rare genital tumors. They are biphasic neoplasms composed of an admixture of malignant epithelial and mesenchymal elements. In descending order of frequency they originate in the uterus, ovaries, fallopian tubes, cervix and vagina. Also they arise denovo from peritoneum. They are highly aggressive and tend to occur in postmenopausal low parity women. Because of rarity, there is as such no treatment guidelines available. Multimodality treatment in the form of radical surgery followed by adjuvant chemotherapy or radiotherapy or combined chemoradiation gives a better prognosis & outcome. Two case reports of such tumors, one from ovary and other from penitoneum are presented along with the review of literature.
Intra-Fetal Laser Ablation of Umbilical Vessels in Acardiac Twin with Success...Apollo Hospitals
This case report describes the successful treatment of an acardiac twin (TRAP sequence) via intra-fetal laser ablation of the umbilical vessels. The patient was a 26 year old pregnant woman at 18 weeks gestation with twins, one normal (Twin A) and one acardiac (Twin B). By 26 weeks, Twin A showed signs of cardiac failure so laser ablation was performed to interrupt blood flow from Twin B to A. This minimally invasive procedure used an Nd: YAG laser to coagulate the vessels under ultrasound guidance. The pregnancy continued successfully, with Twin A delivered via c-section at 35 weeks in good condition. This report demonstrates that intra-fetal laser ablation can safely
Improved Patient Satisfaction At Apollo – A Case StudyApollo Hospitals
1) Indraprastha Apollo Hospital utilized patient satisfaction surveys called Voice of Customer (VOC) tools to identify ways to improve various hospital departments and services.
2) Factors that contributed to an increasing trend in VOC scores over 1.5 years included leadership commitment to quality improvement, improved efficiency, superior clinical care, soft skills enhancement for staff, and improved patient information and complaint resolution.
3) Through consistent efforts such as staff training, improved processes, and addressing issues identified in VOC surveys, Apollo Hospitals achieved higher than target patient satisfaction scores, creating loyal patients with memorable hospital experiences.
Breast Cancer in Young Women and its Impact on Reproductive FunctionApollo Hospitals
Breast cancer is the most common cancer in women in developed countries. Chemotherapy for breast cancer is likely to negatively impact on reproductive function. We review current treatment; effects on reproductive function; breastfeeding and management of menopausal symptoms following breast cancer.
Turner syndrome (gonadal dysgenesis) is one of the most common chromosomal abnormalities occuring 1 in 2500 to 1 in 3000 live-born girls. It is an important cause of short stature in girls and primary amenorrhea in young women that is usually caused by loss of part or all of an X chromosome. This review briefly summarises the current knowledge about the syndrome and the management strategies.
Due to pregnancy thyroid economy is affected with changes in iodine metabolism, TBG and development of maternal goiter. The incidence of hypothyroidism in pregnancy is quite common with autoimmune hypothyroidism being the most important cause. Overt as well as subclinical hypothyroidism has a varied impact on maternal and neonatal outcome. After multiple studies also, routine screening in pregnancy for hypothyroidism can still not be recommended. Management mainly comprises of dosage adjustments as soon as pregnancy is diagnosed based on results of thyroid function tests. The aim should be to keep FT4 at the upper end of normal range.
Growth Hormone Deficiency (GHD) can persist from childhood or be newly acquired. Confirmation through stimulation testing is usually required unless there is a proven genetic/structural lesion persistent from childhood. Growth harmone (GH) therapy offers benefits in body composition, exercise capacity, skeletal integrity, and quality of life measures and is most likely to benefit those patients who have more severe GHD. The risks of GH treatment are low. GH dosing regimens should be individualized. The final decision to treat adults with GHD requires thoughtful clinical judgment with a careful evaluation of the benefits and risks specific to the individual.
Advances in the management of thalassemia have led to marked improvements in the life span and quality of life of children and young adults. This poses new challenges for the treating physicians. There is now increasing recognition that thalassemics have impaired bone health which is multifactorial in etiology. This paper aims to highlight the factors that predispose these patients to osteoporosis and suggests measures to minimise the impact on bone health.
A 34-year-old woman presented with accidental ingestion of mercury that was used in her household to preserve grains. She experienced abdominal radiopaque shadows on X-ray that cleared after two days. Mercury poisoning can result from inhalation, ingestion, or absorption and affects the neurological, gastrointestinal, and renal systems. Diagnosis involves determining exposure history and elevated mercury levels in blood and urine. Supportive treatment includes removal of contaminated materials, irrigation, activated charcoal, chelation agents, and hemodialysis in severe cases.
Laparoscopic Excision of Foregut Duplication Cyst of StomachApollo Hospitals
Retroperitoneal gastric duplication cysts lined by ciliated columnar epithelium are extremely rare lesions and its presentation during adulthood is a diagnostic challenge for treating clinicians. This entity often resembles cystic pancreatic neoplasm, retroperitoneal cystic lesions and sometimes as an adrenal cystic neoplasm. Correct diagnosis on the basis of radiological investigation is difficult and histopathologic analysis. We report a case of gastric duplication cyst in a 16year old girl that mimicked as a retroperitoneal /pancreatic /adrenal cystic lesion and was successfully managed by laparoscopy.
Occupational Blood Borne Infections: Prevention is Better than CureApollo Hospitals
Viral infections like HIV, hepatitis Band C virus pose a big risk to the contacts of individuals with high risk behaviour as well as to the attending health care workers. Blood, semen, vaginal and other potentially infectious materials can transmit the infection to the susceptible contacts. Universal precautions should be strictly implemented during clinical examination, laboratory work and surgical procedures to prevent transmission to the health care providers. Health care workers should receive vaccination for hepatitis B infection. An inadvertent exposure should be managed with proper first aid and infectivity of the source and severity of exposure should be assessed. Severity of exposure is based on the nature and area of exposed surface, mode of injury and volume of infective material. Post-exposure prophylaxis (PEP) should be started as soon as possible after a proper counseling about the effectiveness of post-exposure prophylaxis, side effects and risk of carrying the infection to his familial contacts and its prevention.
Evaluation of Red Cell Hemolysis in Packed Red Cells During Processing and St...Apollo Hospitals
Storage of red cells causes a progressive increase in hemolysis. Inspite of the use of additive solutions for storage and filters for leucoreduction some amount of hemolysis is still inevitable. The extent of hemolysis however should not exceed the permissible threshold for hemolysis even on the 42nd day of storage.
Efficacy and safety of dexamethasone cyclophosphamide pulse therapy in the tr...Apollo Hospitals
Various drugs used to treat pemphigus can cause remission, but none can provide permanent remission as relapses are common. With the introduction of DCP in pemphigus in 1984, patients started being in prolonged/permanent remission. This study was done to compare the efficacy of DCP to oral corticosteroids and cyclophosphamide in combination.
Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS)Apollo Hospitals
This case report describes a 24-year-old man who presented with fever, rash, abdominal pain, and vomiting. He had been taking carbamazepine for seizures. His symptoms and lab results met the criteria for Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS), also known as drug hypersensitivity syndrome. DRESS is caused by certain drugs and is characterized by fever, rash, eosinophilia, and involvement of internal organs like the liver or lungs. Carbamazepine was withdrawn and steroids were started, leading to improvement. The report reviews the characteristics, diagnosis, and treatment of DRESS, noting it is important to identify the causative drug and avoid re-
Difficult Laparoscopic Cholecystectomy-When and Where is the Need to Convert?Apollo Hospitals
Laparoscopic cholecystectomy has now become the treatment of choice for the gall bladder stone. With increasing experience, surgeon has started to take more difficult cases which were considered relative contra indications for laparoscopic removal of gall bladder few years back.
We conducted this study at our hospital and included all laparoscopic cholecystectomy done from May'08 to January'10. Total time taken in surgery, conversion rate and complication rate were analysed. Factors making laparoscopic cholecystectomy difficult were also analysed. We defined difficult laparoscopic cholecystectomy when we found -dense fibrotic adhesions in and around Callot's triangle, gangrenous gall bladder, empyma, large stone impacted at gall bladder neck, contracted gall bladder, Mirrizi's syndrome, h/o biliary pancreatitis, CBD stones, acute cholecystitis of <72 hrs duration.
Out of 206 cases done during above period, 56 cases were considered difficult. Only two cases were converted to open.
With growing experience and technical advancement surgery can be completed in most of the difficult cases. This is important because recently it is shown in literature that laparoscopic cholecystectomy is associated with less morbidity than open method irrespective of duration of the surgery.
Deep vein thrombosis prophylaxis in a tertiary care center: An observational ...Apollo Hospitals
Deep vein thrombosis (DVT) is a major health problem with substantial mortality and morbidity in medically ill patients. Prevention of DVT by risk factor stratification and subsequent antithrombotic prophylaxis in moderate- to severe-risk category patients is the most rational means of reducing morbidity and mortality.
This document describes two cases of unusual manifestations of dengue fever. Case 1 is a 40-year-old man who presented with fever, headache, body aches, and a rash who developed hepatitis, thrombocytopenia, and respiratory distress from dengue hemorrhagic fever. Case 2 is a 24-year-old man who presented with fever and was found to have an intraocular hemorrhage, retinal detachment, ARDS, myocarditis, and hepatitis, also from dengue hemorrhagic fever. The document then reviews atypical neurological and gastrointestinal manifestations that have been reported with dengue infection.
A 71-year-old male presented in ENT department with dysphagia for last three weeks, more to solids than liquids. He had a hard bony bulge in the posterior pharyngeal wall on palpation and hence was referred for an Orthopaedic opinion. Lateral radiograph of the cervical spine revealed diffuse ossification of the anterior longitudinal ligament. This ossification was extending almost half the width of the cervical body from its anterior body at C1 and C2 vertebra level.
This document discusses pediatric liver transplantation. It begins by stating that pediatric liver transplantation is now an established treatment for end-stage liver failure from various causes, with excellent results due to improved immunosuppressive regimens, surgical techniques, and intensive care. It then discusses the historical development of liver transplantation, including the first attempts in the 1960s and key innovations like cyclosporine in the 1980s. The most common indications for pediatric liver transplantation are discussed as extrahepatic biliary atresia and acute liver failure. The document provides an overview of the pre-transplant evaluation process and post-transplant medical management and immunosuppression. It notes that living-related transplantation has helped address the shortage of donor l
Does Over-Masturbation Contribute to Chronic Prostatitis.pptxwalterHu5
In some case, your chronic prostatitis may be related to over-masturbation. Generally, natural medicine Diuretic and Anti-inflammatory Pill can help mee get a cure.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Kat...rightmanforbloodline
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
8 Surprising Reasons To Meditate 40 Minutes A Day That Can Change Your Life.pptxHolistified Wellness
We’re talking about Vedic Meditation, a form of meditation that has been around for at least 5,000 years. Back then, the people who lived in the Indus Valley, now known as India and Pakistan, practised meditation as a fundamental part of daily life. This knowledge that has given us yoga and Ayurveda, was known as Veda, hence the name Vedic. And though there are some written records, the practice has been passed down verbally from generation to generation.
Histololgy of Female Reproductive System.pptxAyeshaZaid1
Dive into an in-depth exploration of the histological structure of female reproductive system with this comprehensive lecture. Presented by Dr. Ayesha Irfan, Assistant Professor of Anatomy, this presentation covers the Gross anatomy and functional histology of the female reproductive organs. Ideal for students, educators, and anyone interested in medical science, this lecture provides clear explanations, detailed diagrams, and valuable insights into female reproductive system. Enhance your knowledge and understanding of this essential aspect of human biology.
Integrating Ayurveda into Parkinson’s Management: A Holistic ApproachAyurveda ForAll
Explore the benefits of combining Ayurveda with conventional Parkinson's treatments. Learn how a holistic approach can manage symptoms, enhance well-being, and balance body energies. Discover the steps to safely integrate Ayurvedic practices into your Parkinson’s care plan, including expert guidance on diet, herbal remedies, and lifestyle modifications.
share - Lions, tigers, AI and health misinformation, oh my!.pptxTina Purnat
• Pitfalls and pivots needed to use AI effectively in public health
• Evidence-based strategies to address health misinformation effectively
• Building trust with communities online and offline
• Equipping health professionals to address questions, concerns and health misinformation
• Assessing risk and mitigating harm from adverse health narratives in communities, health workforce and health system
3. this study, we used the human myeloma cell line RPMI-
8226 as a convenient cellular model system in vitro. We
systematically explored the variables involved with optimi-
zation and standardization of Hoechst 33342 staining vari-
ables such as dye concentration, cell density, staining
duration, staining volume, and mix interval during staining
as well as cell viability prior to flow cytometry (FCM)
analysis.
Importantly, we found that the time interval after cell
subculture and staining is the single most important factor
affecting the SP% and this has not been reported before.
In summary, this study suggests that both the Hoechst stain-
ing and after subculture duration affect the proportion of
SP. Hence, the interpretations of changes in SP% should
be done cautiously. Thus, standardization of these factors
is a reliable platform for drug screening which targets SP.
MATERIALS AND METHODS
Cell culture
The human myeloma cell line RPMI-8226 was obtained
from the Culture Collection (Invitrogen, BioServices India
Pvt Ltd, GIBCOR
, Bangalore). RPMI-8226 cells were
maintained in RPMI-1640 (Invitrogen, BioServices India
Pvt Ltd, GIBCOR
, Bangalore) containing 100 m/mL of
penicillin (Invitrogen), 100 mg/mL of streptomycin (Invitro-
gen), and 10% fetal bovine serum (FBS) (GIBCOR
). Cells
were cultured in T75 or T25 flasks kept in a humidified
incubator with 5% CO2 at 37
C. The cells were seeded
at the density of 0.2 Â 106
cells/mL.
Cell staining
RPMI-8226 cells were harvested after culture for different
periods of time and then stained with Hoechst 33342 dye
(Invitrogen). Briefly, after discarding culture medium, cells
were suspended in the staining medium RPMI-1640þ con-
taining 2% FBS and 10 mM HEPES buffer (Invitrogen).
Live cell number was counted at least twice and adjusted to
a final cell density of 1 Â 106
cells/mL by adding appropriate
volume of warm staining medium. Hoechst 33342 water
solution (1 mg/mL) was then added to make a final concen-
tration of 10 mg/mL followed by incubation in a water bath
at 37
C for 90 min with shaking every 30 min (except in
the experiments that specifically address the shaking factor).
To help gate SP on flow cytometry, samples treated with
1 mg/mL fumitremorgin C (FTC) (Sigma, CITY), an
ABCG2 transporter inhibitor, were included during the entire
staining procedure as controls. Once incubation finished,
samples were immediately put on ice to stop dye efflux.
Subsequently, the cells were centrifuged for 5 min at 300 g
at 4
C and resuspended in 100 mL of ice-cold Hanks’
Balanced Saline Solution (HBSS) (Invitrogen) containing
2% FBS, 100 m/mL of penicillin, 100 mg/mL of strepto-
mycin, and 10 mM HEPES. The samples were kept on ice
before flow cytometry analysis. Propidium iodide (PI)
(Sigma) solution was added at a final concentration of 2 mg/
mL to exclude dead cells just before flow analysis.
Exploration of factors affecting SP%
To explore how cell culture affected SP%, RPMI-8226 cells
were maintainedin culturemedium for a range of time prior to
being harvested for downstream staining. Preparation of the
samples in order to examine the effect of culture time course
on SP% was as follows: RPMI-8226 cells were seeded to four
T25 flasks at 0.2 Â 106
cells/mL under the same conditions.
The cells were cultured for up to 4 days and then harvested
at the end of 1, 2, 3, and 4 days, respectively. Cells were
counted using trypan blue at least twice to adjust same stain-
ing density (1 Â 106
cells/mL) across different experiment
days. Two batches of samples were prepared from different
but close passages of cells. Cells were subsequently stained
undertheconditionsdescribedas below.We alsoinvestigated
the SP% associated with an array of different staining condi-
tions: Hoechst 33342 concentration (ranging from 1e50 mg/
mL), cell density (0.25 Â 106
e2.00 Â 106
cells/mL),
volume (100e2000 mL), duration (30e180 min), shaking
interval (10e90 min), and viable cell proportion
(20e80%). Only one factor was varied and examined each
time, while the others were kept constant. The standard
protocol described before was employed for the staining
procedure except for the testing of the effects of cell viability
prior to FCM analysis on SP%. For those experiments, two
tubes of cell suspensions with a density of 2 Â 106
cells/mL
were prepared in staining medium, one of which was treated
by incubation at 60
C for 20 min until all the cells died. We
then mixed 0.1, 0.2, 0.3, 0.4, 0.5 mL treated cells suspension
with 0.5 mL cell suspension without heat treatment, respec-
tively. The resulting samples with a range of viable cell
percentages were prepared for subsequent staining.
Flow cytometric analysis
Analysiswas performedona four-laserBD LSR II flow cytom-
eter (Bhaskar Medical College, India). The Hoechst 33342 dye
was excited at 355 nm, and its emission fluorescence was
detected using 440/30 nm (Hoechst 33342-Blue) and 670/40
(Hoechst 33342-Red) filter systems. PI was excited at
488 nm, and its fluorescence was detected using a 610/20 nm
filter for dead cell exclusion. The flow data was analyzed.
92 Apollo Medicine 2012 June; Vol. 9, No. 2 Jyothsna
4. RESULTS
This study is based on our previously published results
showing a convincing FCM pattern of stained RPMI-
8226 cells with Hoechst 33342, where a remarkable SP
of about 20% presented with a clear gap existing between
SP and non-SP cells.10
Thus, we decided to observe the
RPMI-8226 myeloma cell line to explore the factors
affecting SP%. The gating strategy used for the present
study. The disappearance of SP cells observed in verapamil
as well as FTC-treated samples was useful for the gating of
SP in flow scattergrams.
Effect of cell culture time course
Previous studies suggested that SP and non-SP cells were in
different cell cycle phases.16
Cell growth in different phases
of the cell cycle would affect SP and non-SP distribution.
To test for this hypothesis, we examined cell culture time
that would affect SP%. Cells subcultured for different
days up to 4 days were used to determine SP% using the
standard staining protocol. The RPMI-8226 cells continued
to grow before reaching the plateau stage on Day 4 after
subculture. Surprisingly, we found that SP% was highest
at Day 1, which was 5.3% and 2.5% for batch 1 and 2,
respectively, and then reduced to 2.3% and 0.4% for batch
1 and 2 at Day 4, respectively.
Effect of Hoechst 33342 dye concentration
Different dye concentrations influenced SP%. Cells were
stained with a range of Hoechst 33342 concentration
from 1 to 50 mg/mL and then assessed for change of SP
%. RPMI-8226 cell was viable in media at low concentra-
tion of Hoechst 33342 stain, but toxic to DNA at high
concentrations. Thus indicated that this DNA-binding dye
was toxic to cells in higher concentrations. The same
gating method was applicable for samples with dye
concentration 20 mg/mL as there was only small change
in flow cytometry profiles. But dye concentration had
a pronounced effect on SP%. A sharp drop of SP% was
observed when dye concentration increased from 7.5 mg/
mL (25.8%) to 10 mg/mL (3.9%), which was followed by
a much gentle reduction to 12.5 mg/mL (0.15%) and
onwards. This significant decrease in SP% at 10 mg/mL
or higher concentrations was not due to the decrease in
the percentage of live cells which were the same at around
75%. Thus, we suggested that the optimum dye concentra-
tion for staining of RPMI-8226 cells to be 10 mg/mL which
was at the junction of the sharp and the gentle slope of SP
% (Fig. 1).
Effect of cell viability
By using a fixed number of live cells of the RPMI-8226,
were determined by SP%. The ratio of live/dead cells
were determined. The mean value of SP% was 3.5% for
samples with 20% live cells, in comparison to SP% of
1.1% when live cell proportion increased to 70%.
Effects of staining conditions
Next, we examined the effects of various components
involved in the actual staining procedure including staining
cell density, volume of staining solution, duration of stain-
ing incubation, interval between mixing during incubation,
and the staining temperature. The results show that all of
these factors contributed changes in SP%.
While examining staining cell density, the viability of
stained cells was stable at around 70% for staining cell
density from 0.5 Â 106
to 2.0 Â 106
cells/mL. No SP cells
were observed at the staining density of 0.75 Â 106
cells/
mL, or lower. The SP% increased sharply when the staining
cell concentration increased to 1.5 Â 106
cells/mL or above.
Varying the duration of staining incubation showed that
there was a constant SP% when the incubation was at least
90 min. Reducing the incubation period resulted in an
increase of SP% while there was no change of viable cell
proportions. Finally, we investigated the effects of the
length of shaking intervals during the incubation using
90 min as the standard incubation period. Fig. 2. no SPs
were detected with frequent shaking of every 15 min or
less, and then SP% proportionally increased in association
with increasing shaking interval time. The result showed
that a frequent mixing of the cells every 15 min may
dramatically result in negative staining results. It is there-
fore important to follow a standard protocol of either a mix-
ing interval of every 30 min or 45 min. In summary, in
order to obtain a consistent SP%, it is important to observe
carefully all the above-mentioned experiment requirements
Fig. 1 Bone marrow stem cells.
Development of cell culture samples for drug Original Article 93
5. of viable cell proportion, dye concentration, staining cell
density, incubation duration, staining volume, and mix
interval, as well as the important factor of the time after
subcultured.
DISCUSSION
It has been known that cancer stem cells (CSCs) resist
current drug therapies and repair DNA after radiation. After
treatment CSCs differentiate more efficiently into daughter
cells. These cells are responsible for the recurrence of
tumors after treatment.17e20
Previous studies showed that
these CSCs survive standard chemotherapeutics in multiple
myeloma.10
It is therefore likely that the only way to cure
cancer is to target against the CSCs.
It has been reported that the number of companies
devoted to research into novel medicines targeting
CSCs.21
In addition, patents covering developments in
CSCs have been increased during these years.21
Although
there are only one or two drugs targeting CSC molecules
currently in Phase II or Phase III/IV clinical trials, there
are thirteen drugs in Phase I trials, with many more in the
development pipeline. All these points lead to the develop-
ment of drugs against CSCs, so that there will be a better
treatment against cancer. This study showed a detailed
affect of different factors influencing SP% in normal or
cancer cells.
In this study, using the human myeloma cell line RPMI-
8226 as a convenient cellular model system in vitro, we
systematically explored multiple factors, namely, Hoechst
33342 dye concentration, cell density, staining duration,
staining volume, mix interval during staining, and cell
viability prior to enumerating SP cells by FCM analysis.
We have highlighted the large variability in SP% which
is obtained when different conditions are used and have
optimized the technical aspects of staining SP cells. With
regards to cell viability, a previous study has also shown
that, for consistent SP staining, the cell count before the
Hoechst staining should include all cells ranging from
dying, dead, to live cells.14
They also suggested that
Hoechst concentration and toxicity curves should be inde-
pendently determined for each new tissue or cell type being
tested.14
In this study, it was found that the time taken after
subculture to staining is an important factor affecting the SP
% which has not been reported previously. Thus, this new
finding should lead to the modification of the preparation of
cell samples for drug screening purposes. Furthermore,
since RPMI-8226 harbors a rich and constant source of
SP cells, it could be used as the positive control for this
platform and for the detailed monitoring of the day-to-day
variation of Hoechst staining performance.
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