This presentation features research on the link between ovarian cancer and hyperglycemia, conducted by Lacey Gibson, a sophomore at Southern Illinois University Carbondale.
Protein purification techniques take advantage of differences in protein properties like charge, size, and binding affinity. The first step is breaking open cells to release proteins. Centrifugation is used to separate subcellular components. Fractionation utilizes differences in solubility, often using ammonium sulfate precipitation. Dialysis removes small solutes by exchanging them through a semipermeable membrane. Column chromatography separates proteins as they migrate through a solid matrix at different rates depending on their interactions. Specific techniques further separate proteins based on ionic charge using ion-exchange, size using size exclusion, or binding affinity using affinity chromatography.
Protein microarrays, ICAT, and HPLC protein purificationRaul Soto
The document discusses the Isotope-Coded Affinity Tag (ICAT) method for protein quantification and identification. ICAT uses chemical labeling reagents that specifically label cysteine residues. There are 4 main steps: 1) Lyse and label protein samples from two states with light and heavy ICAT tags, 2) Mix and proteolyze samples to generate peptide fragments, some tagged, 3) Isolate tagged fragments using avidin affinity chromatography, 4) Analyze isolated peptides using mass spectrometry to identify and quantify proteins between the two states. ICAT allows accurate quantification of complex protein mixtures.
Involvement of multiple P450s and UDP-GTs in the in vitro metabolism of Murag...Luke Lightning
Muraglitazar is metabolized by multiple CYPs including CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. It is also glucuronidated, which is its major metabolic pathway. While the individual contributions of each CYP could not be definitively determined, CYP2C enzymes and CYP3A4 contribute most to oxidation. The extensive involvement of multiple metabolic pathways suggests muraglitazar clearance should be consistent between patients. Some inconsistencies between selective inhibition results and individual CYP experiments may relate to non-specific inhibition.
Metabomeeting2008_rev230408-Jack-parag-final1Shahid Malik
1. The document describes using both 1H-NMR spectroscopy and GC-MS to profile metabolites in human serum and cell extract samples. This combined approach identified over 90 metabolites in a single sample.
2. NMR could identify 53 metabolites in cell extract and 51 in serum, while GC-MS identified 62 metabolites in cell extract and 67 in serum. Some metabolites were uniquely identified by each technique, with 19 identified in both cell extract samples and 24 in both serum samples.
3. The results demonstrate the complementary nature and advantages of NMR and GC-MS for comprehensive metabolite coverage, though NMR has advantages for simultaneous identification and quantification.
Este documento habla sobre la diversidad en el aula y propone estrategias para promover la inclusión y la equidad educativa. Aborda temas como las metodologías, aspectos del proyecto educativo, estrategias de motivación y la integración de contenidos para aceptar las diferencias en apariencia física, género, etnia, orientación sexual, nivel socio-cultural y variedad lingüística. También recomienda construir el conocimiento tomando conciencia de los temores y sentimientos de los estudiantes, y organizar
Este documento habla sobre la diversidad en el aula y propone estrategias para promover la inclusión y la equidad. Aborda temas como las metodologías educativas, las estrategias para integrar contenidos que representen diferentes culturas, géneros y orientaciones sexuales. También recomienda que los docentes tomen conciencia de sus propios prejuicios y aprendan a aceptar las emociones de los estudiantes para crear un ambiente de aprendizaje inclusivo.
Protein purification techniques take advantage of differences in protein properties like charge, size, and binding affinity. The first step is breaking open cells to release proteins. Centrifugation is used to separate subcellular components. Fractionation utilizes differences in solubility, often using ammonium sulfate precipitation. Dialysis removes small solutes by exchanging them through a semipermeable membrane. Column chromatography separates proteins as they migrate through a solid matrix at different rates depending on their interactions. Specific techniques further separate proteins based on ionic charge using ion-exchange, size using size exclusion, or binding affinity using affinity chromatography.
Protein microarrays, ICAT, and HPLC protein purificationRaul Soto
The document discusses the Isotope-Coded Affinity Tag (ICAT) method for protein quantification and identification. ICAT uses chemical labeling reagents that specifically label cysteine residues. There are 4 main steps: 1) Lyse and label protein samples from two states with light and heavy ICAT tags, 2) Mix and proteolyze samples to generate peptide fragments, some tagged, 3) Isolate tagged fragments using avidin affinity chromatography, 4) Analyze isolated peptides using mass spectrometry to identify and quantify proteins between the two states. ICAT allows accurate quantification of complex protein mixtures.
Involvement of multiple P450s and UDP-GTs in the in vitro metabolism of Murag...Luke Lightning
Muraglitazar is metabolized by multiple CYPs including CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. It is also glucuronidated, which is its major metabolic pathway. While the individual contributions of each CYP could not be definitively determined, CYP2C enzymes and CYP3A4 contribute most to oxidation. The extensive involvement of multiple metabolic pathways suggests muraglitazar clearance should be consistent between patients. Some inconsistencies between selective inhibition results and individual CYP experiments may relate to non-specific inhibition.
Metabomeeting2008_rev230408-Jack-parag-final1Shahid Malik
1. The document describes using both 1H-NMR spectroscopy and GC-MS to profile metabolites in human serum and cell extract samples. This combined approach identified over 90 metabolites in a single sample.
2. NMR could identify 53 metabolites in cell extract and 51 in serum, while GC-MS identified 62 metabolites in cell extract and 67 in serum. Some metabolites were uniquely identified by each technique, with 19 identified in both cell extract samples and 24 in both serum samples.
3. The results demonstrate the complementary nature and advantages of NMR and GC-MS for comprehensive metabolite coverage, though NMR has advantages for simultaneous identification and quantification.
Este documento habla sobre la diversidad en el aula y propone estrategias para promover la inclusión y la equidad educativa. Aborda temas como las metodologías, aspectos del proyecto educativo, estrategias de motivación y la integración de contenidos para aceptar las diferencias en apariencia física, género, etnia, orientación sexual, nivel socio-cultural y variedad lingüística. También recomienda construir el conocimiento tomando conciencia de los temores y sentimientos de los estudiantes, y organizar
Este documento habla sobre la diversidad en el aula y propone estrategias para promover la inclusión y la equidad. Aborda temas como las metodologías educativas, las estrategias para integrar contenidos que representen diferentes culturas, géneros y orientaciones sexuales. También recomienda que los docentes tomen conciencia de sus propios prejuicios y aprendan a aceptar las emociones de los estudiantes para crear un ambiente de aprendizaje inclusivo.
Sumberdaya konsumen adalah pendapatan atau kekayaan yang dimiliki konsumen untuk menentukan apa yang akan dibeli. Terdapat tiga jenis sumberdaya konsumen: ekonomi individu, ekonomi keluarga, dan ekonomi rumah tangga. Sumberdaya ekonomi berkaitan dengan pendapatan, sedangkan sumberdaya temporal terkait waktu kerja dan waktu senggang konsumen. Barang dibagi menjadi yang memerlukan waktu dan penghemat
The document provides style recommendations and product descriptions for winter fashion. It features 5 sections that highlight popular winter colors and trends: 1) Winter Grey, 2) Red, 3) Shine, 4) Details, and 5) Military. Each section describes shells or accessories available in that style. The document also promotes the interchangeable shell concept of Miche bags, allowing customers to change a bag's look without changing the entire bag. Product details and prices are listed for shells, handles, and other accessories.
Dokumen tersebut merangkum tentang pengertian dan prosedur Penelitian Tindakan Kelas (PTK). PTK adalah penelitian yang dilakukan guru untuk memperbaiki pembelajaran dengan merancang, melaksanakan, dan merefleksikan tindakan secara kolaboratif. Prosedur PTK umumnya terdiri atas perencanaan, pelaksanaan, pengamatan, dan refleksi yang dilakukan dalam siklus berulang.
Dokumen tersebut membahas upaya peningkatan prestasi belajar Pendidikan Kewarganegaraan (PKn) materi mendeskripsikan Negara Kesatuan Republik Indonesia (NKRI) melalui permainan ular tangga pada siswa kelas VA SDN 01 Manguharjo Madiun tahun pelajaran 2012/2013. Permasalahan yang dihadapi adalah rendahnya aktivitas dan prestasi belajar siswa dalam pembelajaran materi tersebut. Untuk mengatasinya, peneliti mengg
PRINCIPLES AND APPLICATIONS OF CELL VIABILITY ASSAY (MTT ASSAY)Durgadevi Ganesan
The document discusses the principles and applications of MTT cell viability assays. The MTT assay is a colorimetric assay that measures the reduction of MTT by mitochondrial succinate dehydrogenase in metabolically active cells to form an insoluble purple formazan product. The amount of formazan produced is directly proportional to the number of viable cells and can be used to measure cell proliferation, viability, and cytotoxicity in response to drugs or other factors. The MTT assay is inexpensive, rapid, quantitative, and reproducible, making it well-suited for applications like cytotoxicity testing, drug screening, and measuring responses to growth factors.
Cancer is characterized by uncontrolled cell proliferation. Many factors can cause cancer, including external factors like chemicals and radiation, and internal factors like hormones and genetic mutations. While there are 92 approved anticancer drugs, effective therapies are still lacking for many types of cancer. New drugs are needed that are more selective for cancer cells to reduce side effects from long-term treatment. In vitro screening methods are used to identify potential drug candidates, including assays to test cell viability, proliferation, and morphology. Promising candidates then advance to in vivo testing using animal models of cancer like chemically-induced tumors in mice. The goal is to find drugs that can effectively treat cancer while avoiding side effects.
Simvastatin enhances the effects of 5-fluorouracil (5-FU) on human colorectal cancer cell lines. In vitro studies found that pretreatment with simvastatin sensitizes two colorectal cancer cell lines to 5-FU-induced apoptosis in a dose-dependent manner. The combination of simvastatin and 5-FU was more effective at inhibiting cell growth and inducing apoptosis than either treatment alone. This effect involves downregulation of anti-apoptotic proteins Mcl-1 and Bcl-2 as well as increased reactive oxygen species production. Further research is recommended to study the molecular mechanisms and potential in vivo effects.
Chronic myeloid leukemia (CML) is a stem cell disorder caused by the Philadelphia chromosome, which results from the fusion of the BCR gene on chromosome 22 and the ABL gene on chromosome 9. This fusion produces the BCR-ABL protein which exhibits uncontrolled tyrosine kinase activity, driving excessive proliferation of CML cells. CML progresses through chronic, accelerated and blast crisis phases as additional genetic mutations accumulate. Tyrosine kinase inhibitors (TKIs) target the BCR-ABL protein and have significantly improved survival, with a 10-year survival of 85% with TKI therapy. Monitoring response through cytogenetics, FISH and molecular testing guides treatment decisions such as changing or adding other TKIs.
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.
Brentuximab vedotin is an antibody-drug conjugate used to treat Hodgkin lymphoma and systemic anaplastic large cell lymphoma. It consists of the monoclonal antibody cAC10 linked to the microtubule inhibitor MMAE. When the antibody binds to CD30 proteins on lymphoma cells, the complex is internalized and MMAE is released, disrupting the microtubule network and causing cell death. Brentuximab vedotin allows targeted delivery of the cytotoxic agent to minimize side effects compared to conventional chemotherapy. Common side effects include neuropathy, nausea, fatigue, and low blood cell counts.
Brentuximab vedotin is an antibody-drug conjugate used to treat Hodgkin lymphoma and systemic anaplastic large cell lymphoma. It consists of the monoclonal antibody cAC10 linked to the microtubule inhibitor MMAE. When the antibody binds to CD30 proteins on lymphoma cells, the complex is internalized and MMAE is released, disrupting the microtubule network and causing cell death. Brentuximab vedotin allows targeted delivery of the cytotoxic agent to minimize side effects compared to conventional chemotherapy. Common side effects include neuropathy, nausea, fatigue, and low blood cell counts.
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 summarizes a study that investigated the effect of sodium dichloroacetate (DCA) on melanoma cells. The study found that DCA induced apoptosis in melanoma cells in a dose-dependent manner, with higher concentrations of DCA inducing apoptosis more rapidly. Leptin provided a protective effect against DCA-induced apoptosis. The results suggest that targeting cancer cell metabolism by switching them from glycolysis to oxidative phosphorylation through DCA treatment could be a potential therapeutic approach for treating melanoma.
This document provides an overview of myeloid leucopoiesis, or the formation of white blood cells from myeloid progenitor cells. It discusses the sites where white blood cell formation occurs, the growth factors involved in differentiation and proliferation, and the maturation process for granulocytes and monocytes. The key functions of neutrophils, eosinophils, basophils and monocytes/macrophages are also summarized, including their roles in phagocytosis and the innate immune response.
Liposomes are spherical vesicles composed of concentric bilayer membranes made of phospholipids that can encapsulate aqueous solutions. They range in size from 20nm to several micrometers. Liposomes provide advantages for drug delivery such as increased drug efficacy, reduced toxicity, and passive tumor targeting. Common methods for preparing liposomes include physical dispersion, solvent dispersion, and detergent solubilization. Liposomes are evaluated based on properties like size, surface charge, drug encapsulation efficiency, and release kinetics. They have applications in drug delivery, antimicrobial and antiviral therapies, immunology, and cosmetics.
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
Sumberdaya konsumen adalah pendapatan atau kekayaan yang dimiliki konsumen untuk menentukan apa yang akan dibeli. Terdapat tiga jenis sumberdaya konsumen: ekonomi individu, ekonomi keluarga, dan ekonomi rumah tangga. Sumberdaya ekonomi berkaitan dengan pendapatan, sedangkan sumberdaya temporal terkait waktu kerja dan waktu senggang konsumen. Barang dibagi menjadi yang memerlukan waktu dan penghemat
The document provides style recommendations and product descriptions for winter fashion. It features 5 sections that highlight popular winter colors and trends: 1) Winter Grey, 2) Red, 3) Shine, 4) Details, and 5) Military. Each section describes shells or accessories available in that style. The document also promotes the interchangeable shell concept of Miche bags, allowing customers to change a bag's look without changing the entire bag. Product details and prices are listed for shells, handles, and other accessories.
Dokumen tersebut merangkum tentang pengertian dan prosedur Penelitian Tindakan Kelas (PTK). PTK adalah penelitian yang dilakukan guru untuk memperbaiki pembelajaran dengan merancang, melaksanakan, dan merefleksikan tindakan secara kolaboratif. Prosedur PTK umumnya terdiri atas perencanaan, pelaksanaan, pengamatan, dan refleksi yang dilakukan dalam siklus berulang.
Dokumen tersebut membahas upaya peningkatan prestasi belajar Pendidikan Kewarganegaraan (PKn) materi mendeskripsikan Negara Kesatuan Republik Indonesia (NKRI) melalui permainan ular tangga pada siswa kelas VA SDN 01 Manguharjo Madiun tahun pelajaran 2012/2013. Permasalahan yang dihadapi adalah rendahnya aktivitas dan prestasi belajar siswa dalam pembelajaran materi tersebut. Untuk mengatasinya, peneliti mengg
PRINCIPLES AND APPLICATIONS OF CELL VIABILITY ASSAY (MTT ASSAY)Durgadevi Ganesan
The document discusses the principles and applications of MTT cell viability assays. The MTT assay is a colorimetric assay that measures the reduction of MTT by mitochondrial succinate dehydrogenase in metabolically active cells to form an insoluble purple formazan product. The amount of formazan produced is directly proportional to the number of viable cells and can be used to measure cell proliferation, viability, and cytotoxicity in response to drugs or other factors. The MTT assay is inexpensive, rapid, quantitative, and reproducible, making it well-suited for applications like cytotoxicity testing, drug screening, and measuring responses to growth factors.
Cancer is characterized by uncontrolled cell proliferation. Many factors can cause cancer, including external factors like chemicals and radiation, and internal factors like hormones and genetic mutations. While there are 92 approved anticancer drugs, effective therapies are still lacking for many types of cancer. New drugs are needed that are more selective for cancer cells to reduce side effects from long-term treatment. In vitro screening methods are used to identify potential drug candidates, including assays to test cell viability, proliferation, and morphology. Promising candidates then advance to in vivo testing using animal models of cancer like chemically-induced tumors in mice. The goal is to find drugs that can effectively treat cancer while avoiding side effects.
Simvastatin enhances the effects of 5-fluorouracil (5-FU) on human colorectal cancer cell lines. In vitro studies found that pretreatment with simvastatin sensitizes two colorectal cancer cell lines to 5-FU-induced apoptosis in a dose-dependent manner. The combination of simvastatin and 5-FU was more effective at inhibiting cell growth and inducing apoptosis than either treatment alone. This effect involves downregulation of anti-apoptotic proteins Mcl-1 and Bcl-2 as well as increased reactive oxygen species production. Further research is recommended to study the molecular mechanisms and potential in vivo effects.
Chronic myeloid leukemia (CML) is a stem cell disorder caused by the Philadelphia chromosome, which results from the fusion of the BCR gene on chromosome 22 and the ABL gene on chromosome 9. This fusion produces the BCR-ABL protein which exhibits uncontrolled tyrosine kinase activity, driving excessive proliferation of CML cells. CML progresses through chronic, accelerated and blast crisis phases as additional genetic mutations accumulate. Tyrosine kinase inhibitors (TKIs) target the BCR-ABL protein and have significantly improved survival, with a 10-year survival of 85% with TKI therapy. Monitoring response through cytogenetics, FISH and molecular testing guides treatment decisions such as changing or adding other TKIs.
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.
Brentuximab vedotin is an antibody-drug conjugate used to treat Hodgkin lymphoma and systemic anaplastic large cell lymphoma. It consists of the monoclonal antibody cAC10 linked to the microtubule inhibitor MMAE. When the antibody binds to CD30 proteins on lymphoma cells, the complex is internalized and MMAE is released, disrupting the microtubule network and causing cell death. Brentuximab vedotin allows targeted delivery of the cytotoxic agent to minimize side effects compared to conventional chemotherapy. Common side effects include neuropathy, nausea, fatigue, and low blood cell counts.
Brentuximab vedotin is an antibody-drug conjugate used to treat Hodgkin lymphoma and systemic anaplastic large cell lymphoma. It consists of the monoclonal antibody cAC10 linked to the microtubule inhibitor MMAE. When the antibody binds to CD30 proteins on lymphoma cells, the complex is internalized and MMAE is released, disrupting the microtubule network and causing cell death. Brentuximab vedotin allows targeted delivery of the cytotoxic agent to minimize side effects compared to conventional chemotherapy. Common side effects include neuropathy, nausea, fatigue, and low blood cell counts.
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 summarizes a study that investigated the effect of sodium dichloroacetate (DCA) on melanoma cells. The study found that DCA induced apoptosis in melanoma cells in a dose-dependent manner, with higher concentrations of DCA inducing apoptosis more rapidly. Leptin provided a protective effect against DCA-induced apoptosis. The results suggest that targeting cancer cell metabolism by switching them from glycolysis to oxidative phosphorylation through DCA treatment could be a potential therapeutic approach for treating melanoma.
This document provides an overview of myeloid leucopoiesis, or the formation of white blood cells from myeloid progenitor cells. It discusses the sites where white blood cell formation occurs, the growth factors involved in differentiation and proliferation, and the maturation process for granulocytes and monocytes. The key functions of neutrophils, eosinophils, basophils and monocytes/macrophages are also summarized, including their roles in phagocytosis and the innate immune response.
Liposomes are spherical vesicles composed of concentric bilayer membranes made of phospholipids that can encapsulate aqueous solutions. They range in size from 20nm to several micrometers. Liposomes provide advantages for drug delivery such as increased drug efficacy, reduced toxicity, and passive tumor targeting. Common methods for preparing liposomes include physical dispersion, solvent dispersion, and detergent solubilization. Liposomes are evaluated based on properties like size, surface charge, drug encapsulation efficiency, and release kinetics. They have applications in drug delivery, antimicrobial and antiviral therapies, immunology, and cosmetics.
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 MTT assay is a colorimetric assay that uses the yellow tetrazolium dye MTT to measure cellular metabolic activity as a proxy for cell viability. In the assay, viable cells containing NAD(P)H-dependent oxidoreductase enzymes reduce MTT to purple formazan crystals, which are then dissolved and quantified by spectrophotometry. The degree of color formation correlates with the number of viable cells present. The MTT assay is inexpensive, does not require cell transfer, and can be used to assess cell proliferation, cytotoxicity, and apoptosis for a variety of cell types.
Invitro Screening of Anti-Cancer Drugs.Kanthlal SK
The document discusses various in vitro assays that can be used to test potential anti-cancer compounds by examining their effects on tumor cell lines in place of in vivo animal testing. It describes assays such as MTT, membrane integrity dye exclusion, DNA content fluorescent, SRB protein content, and clonogenic assays that analyze cellular properties, viability, proliferation, and colony formation ability. Tumor cell lines from several cancer types can be used with quality control. The MTT and SRB assays are discussed in more detail. In vitro testing allows screening of more compounds using less quantity while reducing animal usage and death.
Cell viability and proliferation assays measure aspects of cellular health and function, such as membrane integrity, metabolic activity, and DNA synthesis. Common assays include MTT, which measures mitochondrial activity; ATP assays, which measure ATP concentration as a marker of viability; Sulforhodamine B, which binds cellular proteins to measure biomass; and propidium iodide staining, which detects compromised membranes. These assays are useful for screening drug toxicity and effects on cell growth.
Presentation on Tamoxifen; prevention and treatment for breast cancer. Prepared for Recent Trends in Therapeutics, CLIN 514, at Humber College, November 2011.
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.
Cell viability assays determine the number of living cells in a culture sample. There are several types of assays, including those measuring enzyme activity, membrane permeability, ATP production, DNA synthesis, and metabolic activity. Metabolic assays use tetrazolium salts or Alamar blue that change color when reduced by living cells, allowing quantification with a spectrophotometer. Membrane integrity assays use dyes like propidium iodide to distinguish living from dead cells based on intact versus compromised membranes.
Role of mitochondrial and hem e function in lung cancer bioenergetics and tum...Mudassir khan
- Lung cancer is the leading cause of cancer deaths worldwide, with non-small cell lung cancer (NSCLC) being the most common type. NSCLC consists of lung adenocarcinoma and lung squamous cell carcinoma.
- Cancer cells rely heavily on metabolic pathways like glycolysis and oxidative phosphorylation (OXPHOS) to generate energy in the form of ATP. Mitochondria play a key role in OXPHOS.
- Studies have shown higher levels of heme, mitochondria, and OXPHOS in NSCLC cells compared to normal cells. When mitochondrial function or heme synthesis is inhibited, it limits cancer cell growth and tumor progression.
Similar to Ovarian cancer and hyperglycemia sigma xi (20)
Role of mitochondrial and hem e function in lung cancer bioenergetics and tum...
Ovarian cancer and hyperglycemia sigma xi
1. OVARIAN CANCER AND
HYPERGLYCEMIA: CAN
METFORMIN BE USED TO HALT
TUMOR GROWTH AND
PROLIFERATION?
LACEY GIBSON
S O PHO M O R E U N D E RG R A D UAT E PR E S ID E N T IA L S C HO LA R
MENTOR: DR. BUCK HALES
DEPARTMENT OF PHYSIOLOGY
SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
2. RESEARCH OBJECTIVES
• Study was undertaken to answer the following question:
“Can Metformin be used to halt growth and proliferation of ovarian cancer?”
• Hypothesis:
• Treatment of cancerous ovary cells lines will lead to an increased buildup of lactic
acid, representing an inability for these cells to produce glucose via
gluconeogenesis, which in turn causes decreased energy reservoir for tumor
proliferation.
• Metformin halts tumor growth and proliferation, as evident by its ability to block
gluconeogenesis.
3. BACKGROUND INFORMATION: WHAT IS
OVARIAN CANCER?
• Ovarian cancer currently has the leading rate of mortality compared with all other
gynecological cancers.
• It is the fifth leading cause of death in women (Ahn et al).
• Largely lacking in methods of early detection and preventative treatment.
4. BACKGROUND INFORMATION: WHAT IS
HYPERGLYCEMIA?
Hyperglycemia = Type II Diabetes = Insulin Resistance
Insulin Resistance:
Insulin released to convert blood glucose to glycogen for
long-term storage in muscle and liver cells
High sugar diet causes high storage of glycogen in cells
Pancreas pumps excess insulin, but insulin receptors do not
respond, causing glucose to remain in blood.
5. BACKGROUND INFORMATION: INSULIN
RESISTANCE AND OVARIAN CANCER
• Excess insulin pumped by pancreas shown by in vitro studies to cause cell
proliferation and prevent apoptosis (Thune et al)
• Excess insulin also affects synthesis of certain hormones, such as estrogen,
which play a role in cell differentiation and proliferation(Thune et al)
• Recent studies have suggested a role for hyperglycemia in the development of a
number of cancers, including endometrial, liver, and pancreatic cancer (Swerdlow
et al)
• Ovarian cancer and hyperglycemia share a variety of risk factors and individuals
diagnosed with hyperglycemia are more likely than by chance to be diagnosed
with cancer (Giovannuci et al.)
6. BACKGROUND INFORMATION: HYPERGLYCEMIC
ENVIRONMENT & OVARIAN CANCER
• Warburg Effect describes ability of fast-growing cancer cells to metabolize glucose
via anaerobic glycolysis in addition to oxidative phosphorylation; More glucose
needed for proliferation (Ladley).
• Hyperglycemia provides a nutrient-rich, growth signal-rich environment for
epithelial ovarian cancer cells, where tumor formation and growth is encouraged
by free radical-induced DNA damage. (Kellenberger et al)
• If hyperglycemia contributes to tumor growth and progression, then anti-diabetes
drugs, such as Metformin, may also have an important antitumor role.
7. BACKGROUND INFORMATION: WHAT IS
METFORMIN?
• 1-carbamimidamido-N,N-dimethylmethanimidamide, C4H11N5
• World’s most popular anti-diabetes drug
• Activates AMP-activated protein kinase in cancer cells, which may play a role in
inhibiting cellular growth by:
• Lowering sugar output from via inhibition of gluconeogenesis
• Ultimately lowering blood sugar
• Starving the cells of their abundant glucose supply that previously allowed them to
proliferate
• Has shown anti-proliferative effects on cancerous tissue from a variety of cell
lines
8. BACKGROUND INFORMATION: WHAT IS LACTIC
ACIDOSIS?
• Lactic acid produced as a bi-product of fermentation, which occurs in cancerous
cells
• Lactic acidosis = overproduction or underutilization (via glucogenesis inhibition)
of lactic acid.
• Biguanide drugs such as Metformin linked to lactic acidosis:
• Decreases gluconeogenesis to lower blood glucose
2 lactate (C3H6O3) + energy (from 16 ATP) ---> glucose (C6H12O6)
• Lactate uptake decreased-> less glucose produced; Cancer cells starved.
Metformin
X
9. BACKGROUND INFORMATION: WHAT IS LACTIC
ACIDOSIS?
• Although rare, when caused by Metformin, lactic acidosis has mortality rate of
50% (Price)
• BUT: 9 cases lactic acidosis/10,000 Metformin users (no difference compared to
placebo); Risk factors include: “age of >60 yr; decreased cardiac, hepatic, or
renal function; diabetic ketoacidosis; surgery; respiratory failure; ethanol
intoxication; and fasting” (Luft)
10. BACKGROUND INFORMATION: GLUCONEOGENESIS: TESTING
THE METFORMIN’S ANTI PROLIFERATIVE MECHANISM
Gluconeogenesis = generation of glucose from noncarbonhydrate carbon substrates
(i.e. lactic acid)
Gluconeogenesis could occur as an additional glucose source for “greedy” ovarian
cancer cells
• Cancer cells undergo fermentation in addition to aerobic respiration (Warburg
Effect)… why not gluconeogenesis too?
How can we test this?
Certain drugs that inhibit cancer cell proliferation should also inhibit gluconeogenesis:
If Metformin inhibits gluconeogenesis, a buildup in lactic acid will occur in
Metformin-treated cell lines.
Lactic Acid
11. METHODS: METFORMIN DOSE-RESPONSE
STUDY
Dose-finding study was performed to determine optimal dose of Metformin for cells
during lactic acid assays:
SKOV3 (cancerous human epithelial ovary) cell lines were treated with media
containing various doses (0-25 mmol/L) of Metformin.
Cell count was measured after 24 hours incubation at 37°C in each treatment.
12. RESULTS: METFORMIN DOSE-RESPONSE STUDY
Total Cell Count
3.5
Total Cells (10^5 cell/mL)
3
• Observations: 2.5
• Cells Treated with 25 mmol/L Metformin 2
Control
appeared smaller and fewer in number after 1.5
10 mmol/L Metformin
second dose, compared to other treatments 1
25 mmol/L Metformin
0.5
• T-25 Cell Counter detected zero control cells
0
after third dose, but cells in flask appeared 1 2 3
to be growing under microscope the next Dose Number
day; Human error in measurement?
• After third treatment of Metformin, there DID
appear to be more cells in group treated
Live Cell Count
100
with 10 mmol/L Metformin than control. 90
80
70
Live Cells (%)
60
50 Control
40
30 10 mmol/L Metformin
20 25 mmol/L Metformin
10
0
1 2 3
Dose Number
13. CONCLUSION: METFORMIN DOSE-RESPONSE
STUDY
• Conclusions:
• Treatment of SKOV3 cells with 10 mmol/L Metformin does not decrease cell
viability
• 10 mmol/L is optimal treatment for cells in Lactic Acid Assays; 25 mmol/L is too
high.
14. METHODS: LACTIC ACID ASSAYS
Lactic Acid levels were tested in Control and Metformin-treated cancerous (SKOV3)
and noncancerous (IOSE) epithelial ovarian cell lines
3 Day Procecedure:
Day 1: Cell lines were passaged and transferred to 96-well plate in normal
media; 24 hours of incubation at 37 C followed.
Day 2: Media was replaced with treatment media (Control or 10 mmol/L
Metformin Hank’s Balanced Salt Solution); 24 hours of incubation at 37 C
followed.
Day 3: Lactic acid production was measured with plate reader.
15. RESULTS: LACTIC ACID ASSAY RESULTS
Results
Control
Metformin Metformin
10 mmol/L 25 mmol/L
Lactic Acid
Avg L- Production, Control vs.
Lactate Metformin-Treated SKOV3
(mM) 0.252 0.61 0.437
SEM 0.02 0.06 0.04
0.8 Cells
Average L-Lactate (mM)
0.6
n 16 16 10 0.4
0.2
***
• Cotrol vs. 10 mmol/L Metformin treatment is 0
significant, p<.001 Control Metformin 10 Metformin 25
mmol/L mmol/L
• Lactic acid production increases with Metformin treatment; 25 Treatment
mmol/L Metformin was too high of a dose for cell
survival, causing decrease in lactic acid production.
16. RESULTS: LACTIC ACID ASSAY RESULTS
Lactic Acid Production in SKOV3 and
IOSE Cell Lines
0.8
0.7
0.6
L-lactate (mM)
0.5
0.4
* *
0.3
0.2
0.1
0
Control SKOV3 Metformin Control IOSE Metformin IOSE
SKOV3
Cell Type
Lactic acid increases with Metformin treatment; Results are significant in cancerous
(SKOV3) cells.
Increase in lactic acid levels also significant in cancerous Metformin-treated cells
compared to noncancerous control-treated cells.
17. CONCLUSION: LACTIC ACID ASSAYS
• Lactic acid levels increased significantly in Metformin-treated cancerous epithelial
ovary cells compared to cancerous cells in control media.
• Increase in lactic acid levels was not significant in noncancerous Metformin-
treated cells compared to noncancerous cells in control media.
• This indicates an inhibition of gluconeogenesis in Metformin-treated cancerous
cells.
18. CONCLUSION: EXPLANATION OF METFORMIN’S EFFECT
ON LACTIC ACID PRODUCTION
(O2 present, returns to) Pyruvate (Gluconeogenisis)
Lactic acid production (Metformin treatment
from fermentation in activates
AMPK, decreasing
addition to aerobic gluconeogenisis)
respiration in cancer
cells Glucose
Lactic Acid Buildup
&
Decreased Glucose Production
Decreased reservoir of glucose for cancer cell proliferation
Increased lactic acid production in Metformin-treated
cells indicates an inhibition of gluconeogenesis, which
indicates an decrease in proliferation of ovarian cancer
cells
19. CONCLUSION: WHERE TO NEXT?
Metformin has the ability to decrease energy for proliferation of ovarian cancer cells
via inhibition of gluconeogenesis. This inhibition of gluconeogenesis is shown by a
decrease in lactic acid levels.
Metformin has potential to be used as a drug to treat patients with ovarian cancer
AND patients with hyperglycemia. Wonder drug!
BUT more testing is needed to confirm cancer-treating abilities…
20. CONCLUSION: FUTURE STUDIES
Future studies:
• Test other intermediaries and enzymes related to gluconeogenesis
• Pyruvate, pyruvate kinase activity, glucose
• In vivo testing of Metformin’s ability to suppress ovarian cancer growth and
proliferation
• Study inflammatory genetic markers (i.e. COX-1 and prostaglandin) in tissue
collected from Metformin-treated & control hens.
21. ACKNOWLEDGEMENTS
I am especially grateful for the knowledge that I have gained by working in Dr. Hales’ lab at
Southern Illinois University of Carbondale. I am thankful for the being allowed to learn in the
presence of all the supportive individuals in his facility. I also truly appreciate the support of
the SIUC’s Saluki Scholar Research Opportunity program, the advice of my father, David
Gibson, and the wisdom of the following authors:
Ahn, Suzie E., Jin Choi, Deivendran Rengaraj, Hee Seo, Whasun Lim, Jae Han, and Gwonhwa Song. "Increased Expression of Cysteine Cathepsins in
Ovarian Tissue from Chickens with Ovarian Cancer." Reproductive Biology and Endocrinology8.1 (2010): 100. Print.
Giovannuci, E., Harlan, D. M., Archer, M. C., Bergenstal, R. M, Gapstur, R. M., Habel, L. A., Pollack, M., Regensteiner, J. G., and Yee, Douglas. “Diabetes
and Cancer: A Consensus Report.” Diabetes Care, 2010, vol. 33, pp. 1674-1685.
Kellenberger, L. D., J. E. Bruin, J. Greenaway, N. E. Campbell, R. A. Moorehead, A. C. Holloway, and J. Petrik. "The Role of Dysregulated Glucose
Metabolism in Epithelial Ovarian Cancer." Journal of Oncology 2010 (2010): 1-13. Print.
Ladley, Sara E. The Role of Metabolic Reorigination and Mitochondria in EOC. Diss. Southern Illinois University Carbondale, 2012. Carbondale, 2012.
Print.
Luft, F. C. "Lactic Acidosis Update for Critical Care Clinicians." Journal of American Society of Nephrology 12.17 (2001): n. pag. Print.
Swerdlow, A. J., S. P. Laing, Z. Qiao, S. D. Slater, A. C. Burden, J. L. Botha, N. R. Waugh, A. D. Morris, W. Gatling, E. A. Gale, C. C. Patterson, and H. Keen.
"Cancer Incidence and Mortality in Patients with Insulin-treated Diabetes: A UK Cohort Study." British Journal of Cancer 92.11 (2005): 2070-075.
Print.
Thune, I. "Sustained Physical Activity, Energy Balance and Risk of Breast Cancer." European Journal of Cancer Prevention 7.Supplement 1 (1998): S67-
68. Print.