The experiment tested the cytotoxic activity of mouse macrophages (RAW264.7 cells) against mouse tumor cells (P815 cells) under different conditions. RAW cells were diluted into 5 samples and mixed with P815 cells in a 96-well plate with or without mediators IFN-gamma, LPS, or both. The highest macrophage cytotoxicity of 13.9% was found for the 5:1 RAW dilution. The most effective mediated cytotoxicity of 4.29% was for RAW cells exposed to both LPS and IFN-gamma. This supported the hypothesis that the combination of mediators would yield the best killing activity. The results demonstrate how macrophage ratios and mediation influence their immune response effectiveness.
This document describes an automated method for yeast two-hybrid screening that pools cDNA library subsets in a liquid format. Key points:
- Yeast strains expressing "bait" proteins are mated with pooled subsets of a cDNA library expressed as "prey" fusions in 96-well plates. Interactors are selected and detected using reporters.
- Testing with simulated libraries containing varying ratios of interacting vs. non-interacting clones showed the method can detect interactions even when the interactor is only 0.1% of the prey pool.
- The method was used to screen two nuclear receptor ligand-binding domains against pooled subsets of two cDNA libraries, recovering both previously known and new interacting proteins.
The document describes several assays for measuring cell viability, cytotoxicity, and apoptosis. It provides details on assay mechanisms and recommended protocols for assays such as CellTiter-Glo, CellTiter 96 Aqueous, CellTiter-Blue, CellTiter-Fluor, CytoTox-Glo, CytoTox-One, Caspase-Glo 9, Caspase-Glo 8, and Caspase-Glo 3/7. Examples of dose-response and time course data generated by the assays on different cell types are also shown.
This study compared the cytotoxicity and induction of genes encoding metabolic enzymes in cells exposed to different alkylated polycyclic aromatic hydrocarbons (PAHs). An SRB assay showed little cell death between exposures of 1uM naphthalene, 1uM dimethyl naphthalene, 1uM BAP, and the vehicle control, except for 5uM BAP. RT-PCR analysis found that 1uM BAP induced expression of CYP1A1 and CYP1B1 genes, but 1uM naphthalene and dimethyl naphthalene did not induce gene expression. Comparing responses to the parent naphthalene and dimethylated naphthalene,
This document summarizes the development and in vitro evaluation of novel sulphonamide compounds as potential antimalarial drugs. Specifically:
1) Novel sulphonamide compounds were designed and synthesized based on the structures of bepotastine and sulphonamides.
2) In vitro screening showed several compounds inhibited the asexual development of Plasmodium falciparum parasites at low micromolar concentrations, particularly during late-stage parasite development.
3) The most potent compounds were further evaluated, with three inhibiting parasite growth below 4 μM, while demonstrating low toxicity to mammalian cells.
Triclosan is an antibacterial agent found in many household products that is studied for its potential mutagenic and toxic effects. The study tested whether triclosan is mutagenic using two E. coli strains in an assay with and without liver enzyme activation. Results showed triclosan did not induce reverse mutations, indicating it is not mutagenic. However, HPLC analysis found new peaks when triclosan was reacted with liver enzymes, suggesting metabolism produces derivatives. The study supports further testing triclosan's effects at ecological concentrations on aquatic life and humans with significant exposure.
This document describes various in vitro models and techniques used to study hepatotoxicity, including hepatocyte cell cultures, viability assays, staining techniques, and molecular analysis methods. Specifically, it discusses using HepG2 and HuH7 cell lines to study hepatotoxicity. It also outlines methods like the Trypan Blue exclusion test, Oil Red O staining, MTT assay, RT-PCR, and Western blotting. Finally, it mentions using fatty acids like palmitic and oleic acids in in vitro models of non-alcoholic fatty liver disease.
This document describes the development of a method to detect human CD4+ T cells specific for influenza A virus using soluble human MHC class II tetramers. Key points:
1) Soluble MHC class II tetramers containing an immunodominant peptide from influenza hemagglutinin were constructed and used to identify antigen-specific T cells from influenza-immune individuals.
2) After 7 days of proliferation in response to stimulation by influenza peptide or vaccine, tetramer-positive cells had undergone extensive division and expressed markers of activated CD4+ T cells.
3) The influenza peptide-specific T cells represented a major subset of proliferating CD4+ T cells responding to whole influenza vaccine.
4)
Clinical isolates of urinary tract infection and candidiasis inhibited by T. ...Diganta Dey
This document summarizes a study that evaluated the antimicrobial activity of extracts from Terminalia arjuna bark. Methanol and water extracts were tested against bacterial and fungal clinical isolates. The methanol extract showed higher total phenolic and flavonoid content. Both extracts inhibited the growth of pathogenic bacteria like S. aureus and fungi like Candida species. Minimum inhibitory concentration values ranged from 0.04-1 mg/mL. Comet assay results also indicated that the extracts induced DNA damage in Candida tropicalis. Overall, the study demonstrated the antibacterial and antifungal properties of T. arjuna bark extracts.
This document describes an automated method for yeast two-hybrid screening that pools cDNA library subsets in a liquid format. Key points:
- Yeast strains expressing "bait" proteins are mated with pooled subsets of a cDNA library expressed as "prey" fusions in 96-well plates. Interactors are selected and detected using reporters.
- Testing with simulated libraries containing varying ratios of interacting vs. non-interacting clones showed the method can detect interactions even when the interactor is only 0.1% of the prey pool.
- The method was used to screen two nuclear receptor ligand-binding domains against pooled subsets of two cDNA libraries, recovering both previously known and new interacting proteins.
The document describes several assays for measuring cell viability, cytotoxicity, and apoptosis. It provides details on assay mechanisms and recommended protocols for assays such as CellTiter-Glo, CellTiter 96 Aqueous, CellTiter-Blue, CellTiter-Fluor, CytoTox-Glo, CytoTox-One, Caspase-Glo 9, Caspase-Glo 8, and Caspase-Glo 3/7. Examples of dose-response and time course data generated by the assays on different cell types are also shown.
This study compared the cytotoxicity and induction of genes encoding metabolic enzymes in cells exposed to different alkylated polycyclic aromatic hydrocarbons (PAHs). An SRB assay showed little cell death between exposures of 1uM naphthalene, 1uM dimethyl naphthalene, 1uM BAP, and the vehicle control, except for 5uM BAP. RT-PCR analysis found that 1uM BAP induced expression of CYP1A1 and CYP1B1 genes, but 1uM naphthalene and dimethyl naphthalene did not induce gene expression. Comparing responses to the parent naphthalene and dimethylated naphthalene,
This document summarizes the development and in vitro evaluation of novel sulphonamide compounds as potential antimalarial drugs. Specifically:
1) Novel sulphonamide compounds were designed and synthesized based on the structures of bepotastine and sulphonamides.
2) In vitro screening showed several compounds inhibited the asexual development of Plasmodium falciparum parasites at low micromolar concentrations, particularly during late-stage parasite development.
3) The most potent compounds were further evaluated, with three inhibiting parasite growth below 4 μM, while demonstrating low toxicity to mammalian cells.
Triclosan is an antibacterial agent found in many household products that is studied for its potential mutagenic and toxic effects. The study tested whether triclosan is mutagenic using two E. coli strains in an assay with and without liver enzyme activation. Results showed triclosan did not induce reverse mutations, indicating it is not mutagenic. However, HPLC analysis found new peaks when triclosan was reacted with liver enzymes, suggesting metabolism produces derivatives. The study supports further testing triclosan's effects at ecological concentrations on aquatic life and humans with significant exposure.
This document describes various in vitro models and techniques used to study hepatotoxicity, including hepatocyte cell cultures, viability assays, staining techniques, and molecular analysis methods. Specifically, it discusses using HepG2 and HuH7 cell lines to study hepatotoxicity. It also outlines methods like the Trypan Blue exclusion test, Oil Red O staining, MTT assay, RT-PCR, and Western blotting. Finally, it mentions using fatty acids like palmitic and oleic acids in in vitro models of non-alcoholic fatty liver disease.
This document describes the development of a method to detect human CD4+ T cells specific for influenza A virus using soluble human MHC class II tetramers. Key points:
1) Soluble MHC class II tetramers containing an immunodominant peptide from influenza hemagglutinin were constructed and used to identify antigen-specific T cells from influenza-immune individuals.
2) After 7 days of proliferation in response to stimulation by influenza peptide or vaccine, tetramer-positive cells had undergone extensive division and expressed markers of activated CD4+ T cells.
3) The influenza peptide-specific T cells represented a major subset of proliferating CD4+ T cells responding to whole influenza vaccine.
4)
Clinical isolates of urinary tract infection and candidiasis inhibited by T. ...Diganta Dey
This document summarizes a study that evaluated the antimicrobial activity of extracts from Terminalia arjuna bark. Methanol and water extracts were tested against bacterial and fungal clinical isolates. The methanol extract showed higher total phenolic and flavonoid content. Both extracts inhibited the growth of pathogenic bacteria like S. aureus and fungi like Candida species. Minimum inhibitory concentration values ranged from 0.04-1 mg/mL. Comet assay results also indicated that the extracts induced DNA damage in Candida tropicalis. Overall, the study demonstrated the antibacterial and antifungal properties of T. arjuna bark extracts.
This study assessed the relative expression levels of five constitutive promoters in E. coli using a lux reporter system. The promoters controlled the lysyl-tRNA synthetase (Lys), ribosomal protein (Spc), twin arginine translocation (Tat), lysine decarboxylase (Ldc), and outer membrane lipoprotein (Lpp) genes. The results showed that the Lpp promoter had the highest expression, followed by Tat, Ldc, Spc, and Lys promoters. This correlates to the importance of these genes in E. coli. All promoters showed a linear relationship between light output and colony forming units. This study provides insights into relative promoter strengths that could inform applications requiring different expression
jove-protocol-51306-using-rna-interference-to-investigate-innate-immune-respo...Brandon S Guthrie
This document summarizes a protocol for using RNA interference (RNAi) to investigate the innate immune response in mouse macrophages. The protocol describes optimizing transfection of two mouse macrophage cell lines, RAW264.7 and J774A.1, using a 96-well nucleofection system. It then details monitoring the effects of RNAi-mediated gene knockdown on the innate immune response after stimulating the macrophages with lipopolysaccharide (LPS). As an example, it discusses using RNAi to inhibit Toll-like receptor family members, which sense LPS and regulate innate immunity.
This document summarizes in vitro experiments evaluating the anti-diabetic effects of alkaloidal fractions from Tinospora cordifolia and pentacyclic acid triterpenoids. Rat insulinoma cells were treated with fractions to measure insulin secretion. The fractions inhibited PTP-1B enzyme activity and glucose production in hepatocytes in a concentration-dependent manner, indicating anti-diabetic effects. Molecular docking suggested the compounds bind to a secondary site on PTP-1B, inhibiting the enzyme by a mixed inhibition mechanism. The results suggest pentacyclic triterpenoids may have potential as insulin sensitizers for treating type 2 diabetes.
This study developed methods to measure immune response in reduced volumes of feline whole blood. Lymphocyte proliferation was measured in response to mitogens like ConA, PHA, and PMA/Ionomycin. Flow cytometry was used to identify lymphocyte populations like CD21+ B-cells, CD5+/CD4+ T-helper cells, and CD5+/CD8+ T-cytotoxic cells in whole blood. Phagocytosis was also successfully measured in whole blood using pHrodo-labeled E. coli bioparticles. These assays were refined to require only 2ml of blood while still obtaining reproducible results, supporting the 3Rs principles of reducing animal use. The methods provide a way to investigate innate
This study examined how cellular cholesterol distribution influences the proteolytic release of the LRP-1 ectodomain in breast cancer cell lines. The researchers found that efficient cholesterol extraction and increased LRP-1 shedding occurred in MDA-MB-231 cells but not MDA-MB-435 cells. Raman microspectroscopy revealed MDA-MB-231 cells distributed cholesterol predominantly intracellularly, while MDA-MB-435 cells distributed it at the plasma membrane. Depleting cholesterol in MDA-MB-231 cells increased LRP-1 shedding by making the substrate more accessible at the cell surface, rather than increasing sheddase enzyme expression. This highlights the relationship between cellular cholesterol distribution and its impact on mod
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.
Screening models for testing of immunological factorsKundlik Rathod
This document discusses screening models for testing immunological factors. It describes both in vitro and in vivo methods. The in vitro methods covered include inhibition of histamine release from mast cells, neutrophil locomotion and chemotaxis assays, and using cell lines like THP-1 monocytes. The in vivo methods covered are using murine models to study humoral antibody response, assessing delayed type hypersensitivity reaction, and measuring macrophage phagocytosis using the carbon clearance test. The goal of these screening models is to test substances that can modulate the immune system.
This research article investigates how plant ribosome-inactivating proteins (RIPs) induce cellular stress responses in human cancer cells. The researchers found that two human cancer cell lines exposed to three RIPs - ricin, riproximin and volkensin - activated the unfolded protein response (UPR), a stress response pathway in the endoplasmic reticulum. This suggests the UPR induction better explains the cellular effects of RIPs, as apoptosis was induced even when some protein translation was still occurring due to ribosomal damage. The study provides new insights into the molecular mechanisms by which RIPs exert their toxic effects on cells.
The document discusses alternatives to animal toxicity testing for phototoxicity and carcinogenicity evaluation. It summarizes the 3T3 neutral red uptake photo toxicity test used to assess phototoxic potential in vitro. It also describes tests to detect genotoxic carcinogens like the Ames assay and mouse lymphoma assay, and non-genotoxic carcinogens using the Syrian hamster embryo cell transformation assay which can identify carcinogens through morphological transformation of cells.
This document summarizes a thesis project aimed at characterizing and developing tools to study the UL133/8 locus of human cytomegalovirus (HCMV). The UL133/8 locus encodes four viral proteins - UL133, UL135, UL136, and UL138 - that are thought to regulate HCMV latency and reactivation. The project cloned the genes for these four proteins and expressed them recombinantly in E. coli to enable future studies using small molecule modulators to probe protein function and interactions. This work provides a foundation for elucidating the mechanism by which these viral proteins control HCMV latency and reactivation, with implications for developing new antiviral therapies.
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
This study evaluated the cytotoxic effects of electronic cigarette vapor (vape) and e-liquid on human bronchial epithelial cells in vitro. Results showed that both vape and e-liquid disrupted tight junctions and increased cytotoxicity in a dose-dependent manner. E-liquid exposure resulted in significantly higher cytotoxicity compared to vape. Additionally, the study found that the highly alkaline pH of vape and e-liquid contributed significantly to cytotoxic effects on cells. This suggests that electronic cigarettes may potentially damage airway epithelial cells and alter lung function with long-term use.
Siavosh Naji-Talakar
[email protected]
TITLE: Enzyme Kinetics
INTRODUCTION
Alkaline phosphatases are enzymes that are typically membrane-bound glycoproteins that catalyze the hydrolytic cleavage of monoesters at basic pH levels2. This enzyme is found in most advanced level eukaryotes and prokaryotes. When the hydrolysis of the monophosphate ester takes place at the basic pH levels an inorganic phosphate is released2. The enzyme can remove phosphate groups from several different types of molecules such as alkaloids, proteins, or nucleotides. In humans’ alkaline phosphatases play critical roles in the growth and development of teeth and bones, however, it can be found it other parts of the body such as in the liver and kidneys3. The phosphatases are essential for mineralization in humans to allow calcium and phosphorus to be deposited in bones and teeth3. The enzyme was reacted with 4-nitrophenyl phosphate as the substrate1. 4-nitrophenyl phosphate does not resemble a protein and is a non-specific substrate commonly used to assay alkaline phosphatases4. When the alkaline phosphatase performs hydrolysis on 4-nitrophenyl phosphate a highly colored phosphate free product is given1,4. This reaction releases the phosphate group on 4-nitrophenyl phosphate to give the product 4-nitrophenolate which has a molar absorptivity at 405nm under basic conditions with an extinction co-efficient of 18.8x103 M-1cm-1 1,4.
To experiment the effects of inhibition on the enzyme the inhibitor phenylalanine at 75mM and Na2HPO4 at 15mM was also used. Inhibition of enzymes may be carried out via irreversible pathways that work through covalent bonds or through reversible pathways. Reversible pathways include a competitive inhibition where the inhibitor binds to the active site of the enzyme or through a noncompetitive pathway where the enzyme binds to a side other than the active site, which may subsequently change the shape or conformation of the active site6. When a phosphate group PO43- is removed by hydrolysis from an organic compound it is referred to as dephosphorylation. This is the reaction in which the phosphatases operate. The reaction is important in a physiological setting because it enables the activation or deactivation of enzymes by removal of phosphoric esters; a prime example is the conversation of adenosine triphosphate to adenosine diphosphate through phosphorylation7.
The Michaelis-Menten kinetics model is used in biochemistry as a model for enzyme kinetics. The model uses an equation to explain the rate of the enzymatic reactions that occur . It does this by relating the reaction rate, Velocity or Vmax, to the substrate concentration, [S]. Vmax describes the constant values for each enzyme-substrate complex with the theoretical maximum velocity of the enzyme to turn over products at maximum saturation of the substrate concentration6. The Km value, known as the Michaelis constant, is the dissociation constant of the enzyme-substrate complex and.
This study investigated the mechanisms by which the HDAC inhibitor vorinostat induces apoptosis in acute myeloid leukemia (AML) cells. The results showed that vorinostat causes early DNA damage and activation of p38 MAPK in AML cell lines. Activation of p38 was required for vorinostat-induced G2/M cell cycle arrest and apoptosis. However, the role of p38 activation varied among different HDAC inhibitors, being pro-apoptotic for vorinostat but not necessary for apoptosis induced by other inhibitors. This highlights the importance of understanding specific mechanisms of individual HDAC inhibitors.
1) Researchers used a technique called Bacterial Enzyme Combinatorial Chemistry (BECC) to create modified forms of LPS with altered lipid A structures. 2) They screened these modified LPS molecules for their ability to activate NF-κB in human macrophage cells compared to naturally occurring pro-inflammatory LPS. 3) They identified one modified LPS molecule, created using the enzymes LpxF and PagP, that was able to strongly compete with and outcompete pro-inflammatory LPS, shifting its EC50 over 400-fold. This molecule shows promise for potentially treating septic shock.
The role of surface charge of ISCOMATRIX nanoparticles on the type of immune ...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
ISCOMATRIX vaccines have now been shown to induce strong antigen-specific cellular or humoral immune responses to a broad range of antigens of viral, bacterial, parasite or tumor. In the present study, we investigated the role of ISCOMATRIX charge in induction of a Th1 type of immune response and protection against Leishmania major infection in BALB/c mice.
Materials and Methods:
Positively and negatively charged ISCOMATRIX were prepared. BALB/C mice were immunized subcutaneously, three times with 2-week intervals, with different ISCOMATRIX formulations. Soluble Leishmania antigens (SLA) were mixed with ISCOMATRIX right before injection. The extent of protection and type of immune response were studied in different groups of mice.
Results:
The group of mice immunized with negatively charged ISCOMATRIX showed smaller footpad swelling upon challenge with L. major and the highest IgG2a production compared with positively charged one. The mice immunized with positively charged ISCOMATRIX showed the lowest splenic parasite burden compared to the other groups. Cytokine assay results indicated that the highest level of IFN- γ and IL-4 secretion was observed in the splenocytes of mice immunized with negatively charged ISCOMATRIX as compared to other groups.
Conclusion:
The results indicated that ISCOMATRIX formulations generate an immune response with mixed Th1/Th2 response that was not protective against challenge against L. major.
- The document examines the role of plasminogen activator inhibitor 1 (PAI-1) in the recruitment of mast cells (MCs) to glioma tumors.
- It finds that neutralizing PAI-1 attenuates the infiltration of MCs into glioma tumors. It also finds that MCs express the PAI-1 receptor LRP1, and blocking LRP1 also attenuates MC migration.
- Activation of the potential PAI-1/LRP1 axis in MCs by purified PAI-1 promotes increased phosphorylation of STAT3 and subsequent MC exocytosis. This indicates the PAI-1/LRP1 axis influences MC recruitment in glioma tumors.
Usage of immunological reactions in diagnostics of infectious diseases. Compl...Eneutron
The document discusses the complement fixation test (CFT), a complex serological reaction used to diagnose infectious diseases. It requires an antigen, antibody, and complement to form an antigen-antibody complex that fixes the complement. A second system using sheep red blood cells and haemolytic serum indicates if complement is fixed. If the antigen and antibody match, complement is fixed and haemolysis does not occur, indicating a positive result. The CFT is used to detect antibodies in diseases like syphilis and rickettsioses. It is more complex than other serological tests but also highly specific and sensitive.
This study compared the effects of three Toll-like receptor (TLR) agonists - P3CSK4 (TLR2 agonist), Lipid A (TLR4 agonist), and Poly I:C (TLR3 agonist) - on human monocytes and dendritic cells. It found that all three TLR agonists enhanced the expansion of IFN-γ producing CD4+ T cells when primed with dendritic cells, but only P3CSK4 and Lipid A enhanced T cell proliferation when primed with monocytes. Distinct molecular signatures induced in monocytes and dendritic cells by each TLR agonist correlated with their differential effects on T cell responses. TNF-α and CXCL
This document summarizes research on the effect of primary metabolites on the production of antibiotics platensimycin and platencin by Streptomyces platensis MA7327. Of 32 primary metabolites tested, including amino acids, vitamins, and nucleic acid derivatives, only L-aspartic acid significantly stimulated antibiotic production. The stimulatory effect of aspartic acid is thought to be due to its role as a precursor in the biosynthesis of the 3-amino-2,4-dihydroxy benzoic acid portion of the platensimycin molecule.
This study assessed the relative expression levels of five constitutive promoters in E. coli using a lux reporter system. The promoters controlled the lysyl-tRNA synthetase (Lys), ribosomal protein (Spc), twin arginine translocation (Tat), lysine decarboxylase (Ldc), and outer membrane lipoprotein (Lpp) genes. The results showed that the Lpp promoter had the highest expression, followed by Tat, Ldc, Spc, and Lys promoters. This correlates to the importance of these genes in E. coli. All promoters showed a linear relationship between light output and colony forming units. This study provides insights into relative promoter strengths that could inform applications requiring different expression
jove-protocol-51306-using-rna-interference-to-investigate-innate-immune-respo...Brandon S Guthrie
This document summarizes a protocol for using RNA interference (RNAi) to investigate the innate immune response in mouse macrophages. The protocol describes optimizing transfection of two mouse macrophage cell lines, RAW264.7 and J774A.1, using a 96-well nucleofection system. It then details monitoring the effects of RNAi-mediated gene knockdown on the innate immune response after stimulating the macrophages with lipopolysaccharide (LPS). As an example, it discusses using RNAi to inhibit Toll-like receptor family members, which sense LPS and regulate innate immunity.
This document summarizes in vitro experiments evaluating the anti-diabetic effects of alkaloidal fractions from Tinospora cordifolia and pentacyclic acid triterpenoids. Rat insulinoma cells were treated with fractions to measure insulin secretion. The fractions inhibited PTP-1B enzyme activity and glucose production in hepatocytes in a concentration-dependent manner, indicating anti-diabetic effects. Molecular docking suggested the compounds bind to a secondary site on PTP-1B, inhibiting the enzyme by a mixed inhibition mechanism. The results suggest pentacyclic triterpenoids may have potential as insulin sensitizers for treating type 2 diabetes.
This study developed methods to measure immune response in reduced volumes of feline whole blood. Lymphocyte proliferation was measured in response to mitogens like ConA, PHA, and PMA/Ionomycin. Flow cytometry was used to identify lymphocyte populations like CD21+ B-cells, CD5+/CD4+ T-helper cells, and CD5+/CD8+ T-cytotoxic cells in whole blood. Phagocytosis was also successfully measured in whole blood using pHrodo-labeled E. coli bioparticles. These assays were refined to require only 2ml of blood while still obtaining reproducible results, supporting the 3Rs principles of reducing animal use. The methods provide a way to investigate innate
This study examined how cellular cholesterol distribution influences the proteolytic release of the LRP-1 ectodomain in breast cancer cell lines. The researchers found that efficient cholesterol extraction and increased LRP-1 shedding occurred in MDA-MB-231 cells but not MDA-MB-435 cells. Raman microspectroscopy revealed MDA-MB-231 cells distributed cholesterol predominantly intracellularly, while MDA-MB-435 cells distributed it at the plasma membrane. Depleting cholesterol in MDA-MB-231 cells increased LRP-1 shedding by making the substrate more accessible at the cell surface, rather than increasing sheddase enzyme expression. This highlights the relationship between cellular cholesterol distribution and its impact on mod
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.
Screening models for testing of immunological factorsKundlik Rathod
This document discusses screening models for testing immunological factors. It describes both in vitro and in vivo methods. The in vitro methods covered include inhibition of histamine release from mast cells, neutrophil locomotion and chemotaxis assays, and using cell lines like THP-1 monocytes. The in vivo methods covered are using murine models to study humoral antibody response, assessing delayed type hypersensitivity reaction, and measuring macrophage phagocytosis using the carbon clearance test. The goal of these screening models is to test substances that can modulate the immune system.
This research article investigates how plant ribosome-inactivating proteins (RIPs) induce cellular stress responses in human cancer cells. The researchers found that two human cancer cell lines exposed to three RIPs - ricin, riproximin and volkensin - activated the unfolded protein response (UPR), a stress response pathway in the endoplasmic reticulum. This suggests the UPR induction better explains the cellular effects of RIPs, as apoptosis was induced even when some protein translation was still occurring due to ribosomal damage. The study provides new insights into the molecular mechanisms by which RIPs exert their toxic effects on cells.
The document discusses alternatives to animal toxicity testing for phototoxicity and carcinogenicity evaluation. It summarizes the 3T3 neutral red uptake photo toxicity test used to assess phototoxic potential in vitro. It also describes tests to detect genotoxic carcinogens like the Ames assay and mouse lymphoma assay, and non-genotoxic carcinogens using the Syrian hamster embryo cell transformation assay which can identify carcinogens through morphological transformation of cells.
This document summarizes a thesis project aimed at characterizing and developing tools to study the UL133/8 locus of human cytomegalovirus (HCMV). The UL133/8 locus encodes four viral proteins - UL133, UL135, UL136, and UL138 - that are thought to regulate HCMV latency and reactivation. The project cloned the genes for these four proteins and expressed them recombinantly in E. coli to enable future studies using small molecule modulators to probe protein function and interactions. This work provides a foundation for elucidating the mechanism by which these viral proteins control HCMV latency and reactivation, with implications for developing new antiviral therapies.
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
This study evaluated the cytotoxic effects of electronic cigarette vapor (vape) and e-liquid on human bronchial epithelial cells in vitro. Results showed that both vape and e-liquid disrupted tight junctions and increased cytotoxicity in a dose-dependent manner. E-liquid exposure resulted in significantly higher cytotoxicity compared to vape. Additionally, the study found that the highly alkaline pH of vape and e-liquid contributed significantly to cytotoxic effects on cells. This suggests that electronic cigarettes may potentially damage airway epithelial cells and alter lung function with long-term use.
Siavosh Naji-Talakar
[email protected]
TITLE: Enzyme Kinetics
INTRODUCTION
Alkaline phosphatases are enzymes that are typically membrane-bound glycoproteins that catalyze the hydrolytic cleavage of monoesters at basic pH levels2. This enzyme is found in most advanced level eukaryotes and prokaryotes. When the hydrolysis of the monophosphate ester takes place at the basic pH levels an inorganic phosphate is released2. The enzyme can remove phosphate groups from several different types of molecules such as alkaloids, proteins, or nucleotides. In humans’ alkaline phosphatases play critical roles in the growth and development of teeth and bones, however, it can be found it other parts of the body such as in the liver and kidneys3. The phosphatases are essential for mineralization in humans to allow calcium and phosphorus to be deposited in bones and teeth3. The enzyme was reacted with 4-nitrophenyl phosphate as the substrate1. 4-nitrophenyl phosphate does not resemble a protein and is a non-specific substrate commonly used to assay alkaline phosphatases4. When the alkaline phosphatase performs hydrolysis on 4-nitrophenyl phosphate a highly colored phosphate free product is given1,4. This reaction releases the phosphate group on 4-nitrophenyl phosphate to give the product 4-nitrophenolate which has a molar absorptivity at 405nm under basic conditions with an extinction co-efficient of 18.8x103 M-1cm-1 1,4.
To experiment the effects of inhibition on the enzyme the inhibitor phenylalanine at 75mM and Na2HPO4 at 15mM was also used. Inhibition of enzymes may be carried out via irreversible pathways that work through covalent bonds or through reversible pathways. Reversible pathways include a competitive inhibition where the inhibitor binds to the active site of the enzyme or through a noncompetitive pathway where the enzyme binds to a side other than the active site, which may subsequently change the shape or conformation of the active site6. When a phosphate group PO43- is removed by hydrolysis from an organic compound it is referred to as dephosphorylation. This is the reaction in which the phosphatases operate. The reaction is important in a physiological setting because it enables the activation or deactivation of enzymes by removal of phosphoric esters; a prime example is the conversation of adenosine triphosphate to adenosine diphosphate through phosphorylation7.
The Michaelis-Menten kinetics model is used in biochemistry as a model for enzyme kinetics. The model uses an equation to explain the rate of the enzymatic reactions that occur . It does this by relating the reaction rate, Velocity or Vmax, to the substrate concentration, [S]. Vmax describes the constant values for each enzyme-substrate complex with the theoretical maximum velocity of the enzyme to turn over products at maximum saturation of the substrate concentration6. The Km value, known as the Michaelis constant, is the dissociation constant of the enzyme-substrate complex and.
This study investigated the mechanisms by which the HDAC inhibitor vorinostat induces apoptosis in acute myeloid leukemia (AML) cells. The results showed that vorinostat causes early DNA damage and activation of p38 MAPK in AML cell lines. Activation of p38 was required for vorinostat-induced G2/M cell cycle arrest and apoptosis. However, the role of p38 activation varied among different HDAC inhibitors, being pro-apoptotic for vorinostat but not necessary for apoptosis induced by other inhibitors. This highlights the importance of understanding specific mechanisms of individual HDAC inhibitors.
1) Researchers used a technique called Bacterial Enzyme Combinatorial Chemistry (BECC) to create modified forms of LPS with altered lipid A structures. 2) They screened these modified LPS molecules for their ability to activate NF-κB in human macrophage cells compared to naturally occurring pro-inflammatory LPS. 3) They identified one modified LPS molecule, created using the enzymes LpxF and PagP, that was able to strongly compete with and outcompete pro-inflammatory LPS, shifting its EC50 over 400-fold. This molecule shows promise for potentially treating septic shock.
The role of surface charge of ISCOMATRIX nanoparticles on the type of immune ...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
ISCOMATRIX vaccines have now been shown to induce strong antigen-specific cellular or humoral immune responses to a broad range of antigens of viral, bacterial, parasite or tumor. In the present study, we investigated the role of ISCOMATRIX charge in induction of a Th1 type of immune response and protection against Leishmania major infection in BALB/c mice.
Materials and Methods:
Positively and negatively charged ISCOMATRIX were prepared. BALB/C mice were immunized subcutaneously, three times with 2-week intervals, with different ISCOMATRIX formulations. Soluble Leishmania antigens (SLA) were mixed with ISCOMATRIX right before injection. The extent of protection and type of immune response were studied in different groups of mice.
Results:
The group of mice immunized with negatively charged ISCOMATRIX showed smaller footpad swelling upon challenge with L. major and the highest IgG2a production compared with positively charged one. The mice immunized with positively charged ISCOMATRIX showed the lowest splenic parasite burden compared to the other groups. Cytokine assay results indicated that the highest level of IFN- γ and IL-4 secretion was observed in the splenocytes of mice immunized with negatively charged ISCOMATRIX as compared to other groups.
Conclusion:
The results indicated that ISCOMATRIX formulations generate an immune response with mixed Th1/Th2 response that was not protective against challenge against L. major.
- The document examines the role of plasminogen activator inhibitor 1 (PAI-1) in the recruitment of mast cells (MCs) to glioma tumors.
- It finds that neutralizing PAI-1 attenuates the infiltration of MCs into glioma tumors. It also finds that MCs express the PAI-1 receptor LRP1, and blocking LRP1 also attenuates MC migration.
- Activation of the potential PAI-1/LRP1 axis in MCs by purified PAI-1 promotes increased phosphorylation of STAT3 and subsequent MC exocytosis. This indicates the PAI-1/LRP1 axis influences MC recruitment in glioma tumors.
Usage of immunological reactions in diagnostics of infectious diseases. Compl...Eneutron
The document discusses the complement fixation test (CFT), a complex serological reaction used to diagnose infectious diseases. It requires an antigen, antibody, and complement to form an antigen-antibody complex that fixes the complement. A second system using sheep red blood cells and haemolytic serum indicates if complement is fixed. If the antigen and antibody match, complement is fixed and haemolysis does not occur, indicating a positive result. The CFT is used to detect antibodies in diseases like syphilis and rickettsioses. It is more complex than other serological tests but also highly specific and sensitive.
This study compared the effects of three Toll-like receptor (TLR) agonists - P3CSK4 (TLR2 agonist), Lipid A (TLR4 agonist), and Poly I:C (TLR3 agonist) - on human monocytes and dendritic cells. It found that all three TLR agonists enhanced the expansion of IFN-γ producing CD4+ T cells when primed with dendritic cells, but only P3CSK4 and Lipid A enhanced T cell proliferation when primed with monocytes. Distinct molecular signatures induced in monocytes and dendritic cells by each TLR agonist correlated with their differential effects on T cell responses. TNF-α and CXCL
This document summarizes research on the effect of primary metabolites on the production of antibiotics platensimycin and platencin by Streptomyces platensis MA7327. Of 32 primary metabolites tested, including amino acids, vitamins, and nucleic acid derivatives, only L-aspartic acid significantly stimulated antibiotic production. The stimulatory effect of aspartic acid is thought to be due to its role as a precursor in the biosynthesis of the 3-amino-2,4-dihydroxy benzoic acid portion of the platensimycin molecule.
Similar to 475_LabReport_MpgCytotoxicity.docx (20)
1. Cytotoxicity in Mouse Macrophage RAW264.7 cells against Mouse Mastocytoma P815 cells,
Mediated by IFN-alpha, LPS, or Both
Anastasia Belyakova
February 24th, 2016
Abstract
2. The immunological response of macrophages upon pathogenic attack is important in the
survival of an organism. The purpose of this experiment series was to analyze and quantify the
cytotoxic activity of the mouse macrophage cell line (RAW264.7) upon exposure to mouse
mastocytoma tumor cells (P815) as a model of the innate immune response mechanism by measuring
LDH release by lysed cells. To elucidate the effectiveness of the macrophage reaction, we diluted a
concentrated RAW cell solution into five samples, and prepared a 96 well plate to which we added a
constant amount of P815 cells, as well as mediators IFN-gamma, LPS, or both in order to determine
what combination would yield the best LDH release. Once the absorbances were run, cytotoxicity
could be calculated. We found that the highest percentage of cytotoxicity was for the 5:1 (RAW #2)
dilution at 13.9%, and the most effective cytotoxicity in the 3:1 RAW #5 mediator-affected killing
activity was for the co-culture with both LPS and IFN-gamma present in solution at 4.29%.
Introduction
The immune system, though complex, has a straightforward set of functions: to recognize
foreign infectious particles and invoke a systemic response to eradicate them. In order to survive, an
individual must have functional innate and adaptive immune response mechanisms if the body's
defense barriers are unsuccessful at preventing pathogenic entry. The innate response is termed “natural
resistance” because it is fast and non-specific – as long as the effector cells recognize invading cells as
non-self (Schwander, 6). Macrophages are the cells that are involved in antigen recognition and
cytokine signaling to start the immune response cycle once the foreign particles breach barriers and
invade host tissue.
Once the macrophage comes into contact with a foreign particle, it performs a pattern
recognition. The surface of many pathogenic microorganisms have a conserved pattern of molecular
structures that are not present on the host's cells, and macrophages have pattern recognition receptors
(PRR) that are used to recognize the pathogen-associated molecular patterns (PAMPS) in order to then
3. enact the proper systemic response of secreting cytokines and chemokines (Janeway, 11-12). Then
once the inflammatory response has been induced, the macrophages, along with other inflammatory
cells, engulf and lyse the target foreign cells, thereby eradicating the infection. In order to test the
effectiveness of the macrophages against invading cells, a CytoTox 96 Non-Radioactive Cytotoxicity
Assay can be performed. This method colorimetrically measures lactate dehydrogenase (LDH) released
by lysed target cells, with which we can quantitatively measure the cytotoxic activity of the
macrophages (Schwander, 18). Cytotoxic activity is not only induced by the presence of pathogens, but
can be mediated as well. In our experiment, we looked the effect of LPS and IFN-gamma, and
compared their effect on the macrophage activity by themselves and in combination. Our hypothesis
was that the highest effector:target cell ratio would have the most effective cytotoxic activity, and that
the combination of LPS and IFN-gamma with the RAW #5 3:1 ratio would also have the highest killing
activity as opposed to lacking mediators or having only one in solution.
Materials and Methods
In the series of experiments performed to understand the role of macrophages, it was important
to observe their phagocytic ability. Upon extracting macrophages and lymphocytes (peritoneal exudate
cells, PEC) out of the peritoneum of a mouse and exposing them to latex pellets, we prepared Petri
dishes and looked at the macrophage reaction to the foreign particles. As expected, the macrophages
adhered to the pellets. Then in the second experiment of the macrophage series, we prepared the
macrophage dilutions. We used a cell culture medium composed of 5ml DMEM/F12 (without
indicator) and 3% fetal calf serum in a 15ml conical tube. The stock concentration of RAW264.7 cells
was [2.04 x 106 cells/ml], which we diluted into five eppendorf tubes (#1-5) with tube RAW #1 as the
starting macrophage concentration, tube RAW #2 was diluted to [2.4 x 105 cells/ml], tubes RAW #3
and RAW #5 diluted to [1.03 x 106 cells/ml] and tube RAW #4 diluted to [2.67 x 104 cells/ml]. Once
the dilutions were prepared and mixed, 100 ul of each dilution were added to their respective wells on
4. the 96 microtiter well plate, and the mediators LPS and IFN-gamma were added to respective wells
before incubating for an hour at 37 degrees Celsius. After incubation, 100 ul of P815 target cells were
added to the wells. The ratio of effector to target cells for the five concentrations were 10:1, 5:1, 3:1,
1:1 and 3:1 for RAW #1-5, respectively. The well plate was again incubated for approximately sixteen
hours before the addition of 20ul 10X lysis solution to determine the max lyses, and the plates were
centrifuged at 1200 rpm for five minutes, in order to release the LDH into the supernatant (and leave
irrelevent particles coagulated at the bottom. After this, 50ul of supernatant solutions were added to a
new well plate and frozen at -20 Celsius to hinder the progress of a reaction.
In the final experiment, the well plate was prepared for analysis of macrophage killing. First
50ul of the positive control of LDH at 1:10,000 dilution was added to the A1 well to use as reference
for the rest of the plate. Then 50ul of substrate mix was added to all the wells in the assay, and
incubated in a dark, room temperature environment for half an hour. To end the lysis reaction, 50ul of
stop solution was then added to all the wells and absorbance of the well plate was measured and
recorded at an absorbance of 490nm. Then the absorbance values were recorded and cytotoxicity
results were calculated and compared.
Results
Table 1: Absorbance values of 96 microtiter plate quantitative measurement of LDH release
1 3 4 5 6 8 9 10 11
A 0.7982 0.5678 1.5949 0.6036
B 1.4523 1.21
C 0.1052 0.1161
D 0.2148 0.1915
E 0.3862 0.2599 0.2073 0.1446 0.2779 0.213 0.2069 0.2579
5. F 0.47 0.3438 0.2932 0.2277 0.3146 1.1888 0.1406 0.1942
G 0.5785 0.6747 0.3838 0.2451 0.3257 0.3207 0.4564 0.4803
Table 1 shows the raw data of absorbance values measured by the Cytotox 96 Non-Radioactive
Cytotoxicity Assay, measured at 490nm. Well A1 is the positive control LDH reference to which all
other LDH release absorbances are compared.
Table 2: Percent Cytotoxicity Values for RAW #1-5 and mediated RAW #5
RAW Effector:Target % Cytotoxicity
#1 10:1 4.04
#2 5:1 13.9
#3 3:1 3.43
#4 1:1 -0.06
#5 (no mediator) 3:1 0.47
#5 LPS(+) 3:1 -9.88
#5 IFN-gamma(+) 3:1 2.18
#5 LPS(+) IFN-gamma(+) 3:1 4.29
Table 2 shows the comparison of cytotoxicity among the RAW264.7 dilutions #1-4 and variably
mediated RAW #5 wells. The highest cytotoxicity values were for RAW #2 at 13.9%, and at RAW #5
LPS(+) and IFN-gamma(+).
Discussion
Understanding how the immune system functions correctly is imperative to dealing with a
compromised immune system. In this series of experiments, we tested and analyzed the cytotoxic
effects of macrophages upon exposure to pathogenic cells, and compared cytotoxic effectiveness of the
effector cells in different ratios to target cells, as well as under mediation. What was interesting to
observe was the highest (10:1) effector to target cell ratio did not correspond to the prediction that the
6. cytotoxicity percentage would be highest. In fact the 5:1 ratio produced the best result. The reasoning
for this may be because the target cells were so much outnumbered, they were lysed, and the effector
cells for the most part were not – whereas in the other ratios where target cells were higher in ratio,
there was more of an LDH release and therefore a change in cytotoxic effect. In the 5:1 ratio, the
%cytotocity was at 13.9%, as compared to the 4.04% with the 10:1 ratio. However, with a lower ratio,
there is clearly more of a strain on the RAW264.7 cells because there are more P815 cells to attack, and
inevitably that may cause more effector cell death as well. This is evident in the 1:1 ratio, where the
cytotoxicity is in fact -0.06%, showing that the effector cells struggled to fight the infection.
Alternatively, the mediated RAW #5 cell solutions behaved according to original predictions –
the co-culture combination of both LPS and IFN-gamma mediators produced the highest cytotoxic
percentage result at 4.29%. Surprisingly, the lowest cytotoxic effect was for the LPS(+) solution, which
resulted in a -9.88% cytotoxic effect – although LPS stimulates macrophage activity, the lack of IFN-
gamma slowed down the positive feedback of activating the macrophages fast enough.
The understanding of beneficial effector to target cell ratios and mediation helps in explaining
why the immune response is so important. As we can see, the earlier the immune cells recognize a
pathogen and respond to it, the less cytotoxic effect there is and the faster the infection is eradicated.
Due to the effectiveness of the macrophages in their role of recognition and inflammatory stimulation,
organisms are given a chance to overcome infections and survive.
References:
Covey, L., Firestein, B., Grudzien, E., Schwander, M., Xie, P., Immunology Laboratory Manual.
Rutgers University, Department of Cell Biology & Neuroscience 2016:6-18pp.
Murphy KM, P Travers, M Walport (Eds.) (2010) Janeway's Immunobiology. 8th Edition. New
York:Taylor & Francis, Inc. 11-12pp.