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Viablity assay
- 1. DEPARTMENT OF IMMUNOLOGYANDMOLECULAR BIOLOGY VIABLITY ASSAY By :- Birhanu Ayelign 1 viablity assay UNIVERSITY OF GONDAR COLLEGE OF MEDICINE AND HEALTH SCIENCES SCHOOL OF BIOMEDICALAND LABORATORY SCIENCES 5/29/2017
- 2. Objective At the endof this portion you will be able to :- Define viability assay? What are the classification of viability assay? What are the application of viability assay? Discuses some of the common viability assay? 5/29/2017 2viablity assay
- 3. Introduction To ViabilityAssay A viability assay • is an assay to determine viability of organism or cell • Viability can be distinguished live to died cell 5/29/2017 viablity assay 3
- 4. Features of viablecell • They are based on various cell functions such as – enzyme activity, – cell membrane permeability, – cell adherence, – ATP production, – co-enzyme production, and – nucleotide uptake activity. 5/29/2017 viablity assay 4
- 5. Introduction To ViabilityAssay con… Used to measure a • markers that indicate the number of – dead cells (cytotoxicity assay), – the number of live cells(viability assay), • the total number of cells or • the mechanism of cell death (e.g., apoptosis). 5/29/2017 viablity assay 5
- 6. Application of viabilityassay • To detect cytotoxic or growth inhibitory lymphokines • To detect mammalian cell survival and proliferations • To diagnose disease • To diagnose male infertility • To screen drugs 5/29/2017 viablity assay 6
- 7. Classification of viabilityassays A. Cytolysis or membrane leakage assays B. Mitochondrial activity or caspase assays C. Functional assays D. Genomic and proteomic assays 5/29/2017 viablity assay 7
- 8. Common methods usedin viablity assay MTT cell viability assay WST methods Trypan Blue Lactate dehydrogenase (LDH) Flow cytometry PCR ??????????? 5/29/2017 viablity assay 8
- 9. MTT CELL VIABILITYASSAY •3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide • Application – screening a large numbers of cytotoxicity of chemicals. – cancer chemotherapy ( anti- cancer drugs) – Sperm analysis – Cell proliferation 5/29/2017 viablity assay 9
- 10. Principles of MTTviablity assay 5/29/2017 viablity assay 10
- 11. Procedure for MTTassay 1. Add 100 ml cell in to microplate 2. add 10 ml WTT reagent solution 3. incubated for 3 hr at 37 °C. 4. Subsequently, added 100 ml DMSO to dissolve the resulting formazan by sonication 5. Absorbance was measured at 570 nm using a microplate reader. 6. absorbance is proportional to concentration of live cell 5/29/2017 viablity assay 11
- 12. Sperm MTT assay 5/29/2017viablity assay 12
- 13. water-soluble tetrazolium saltcolorimetric cell viability assay • 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5- (2,4-disulfophenyl)-2H tetrazolium, monosodium salt • Application – screening a large numbers of cytotoxicity of chemicals. – cancer chemotherapy ( anti- cancer drugs) 5/29/2017 viablity assay 13
- 14. WST assay procedure 1.Add 100 ml cell in to microplate 2. add 10 ml WST reagent solution 3. incubated for 2 h at 37 °C. 4. Absorbance was measured at 450 nm using a microplate reader. 5. the absorbance is proportional to concentration of live cell 5/29/2017 viablity assay 14
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- 16. Trypan Blue • widelyused assay for staining dead cells ( blue color) • viable cell must unstained cells • number of cell colonies are counted using a microscope as a cell viability indicator 5/29/2017 viablity assay 16
- 17. Cell viablity, appoptosisand necrosis assay 5/29/2017 viablity assay 17
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- 19. ACKNOWLEGMENT I would liketo express my deepest and heartfelt gratitude to our instructor Mr. Demeke G. & Mr. Fitsumbrhan Tajebew give me an opportunity to do this assignment . Finally, I would like to thank immunology and molecular biology, school of biomedical and laboratory science, Gondar university. 5/29/2017 viablity assay 19
- 20. Reference I. Green LM,Reade JL, Ware CF. Rapid colormetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines. Journal of immunological methods. 1984 May 25;70(2):257-68. II. Molecular genetics of bacteria: Jermey W.Dale & Simone F. Park, 5th edition. III. Geneomes : T.A. Br own, 2th edition. viablity assay 205/29/2017
- 21. 5/29/2017 viablity assay21 Thank u for Your Attention
Editor's Notes
- #7 OFT,,,, hereditary spherocytosis To maintain viable cells in vivo/vitro the cells must be suspended in a solution with an osmotic concentration relatively equal to their interior osmotic concentration: ISOTONIC :0.85% NaCl solution. T. pallidium,,,, immobilization test LDH,,,,, myocardiac infraction, hemolysis, liver disease Male fertility by sperm analysis Use Premixed WST-1 to measure cell proliferation and viability, or to measure cytotoxicity and growth inhibition. A mitochondrial enzyme in viable cells converts WST-1 to a dye…simply measure its absorbance.When cells are proliferating, dye accumulates. When cells are not proliferating, dye levels drop.
- #8 Cytolysis or membrane leakage assays: This category includes the lactate dehydrogenase assay, a stable enzyme common in all cells which can be readily detected when cell membranes are no longer intact. Examples:Propidium iodide,Trypan blue7-Aminoactinomycin D Mitochondrial activity or caspase assays: Resazurin and Formazan (MTT/XTT) can assay for various stages in the apoptosis process that foreshadow cell death. Functional assays: Assays of cell function will be highly specific to the types of cells being assayed. For example, motility is a widely used assay of sperm cell function. Gamete survival can be used to assay fertility, in general. Red blood cells have been assayed in terms of deformability, osmotic fragility, hemolysis, ATP level, and hemoglobin content. For transplantable whole organs the ultimate assay is the ability to sustain life after transplantation, an assay which is not helpful in preventing transplantation of non-functional organs. Genomic and proteomic assays: Cells can be assayed for activation of stress pathways using DNA microarrays and protein chips.
- #9 In order to address this limitation, alternative PCR-based strategies have been developed. This article reviews two complementary strategies. One strategy, termed viability PCR, or vPCR, correlates viability with cell envelope impermeability (9, 10). In viability PCR, microbes in samples are incubated with a membrane-impermeative reagent such as propidium monoazide (PMA). Upon photoactivation, PMA binds tightly to exposed DNA and interferes with PCR amplification. Nonviable cells with damaged membranes, and free nucleic acids, are not protected from the reagent, and their amplification is inhibited after the reagent-DNA complex is photoactivated. In contrast, viable cells with intact cell membranes exclude PMA, enabling strong quantitative PCR (qPCR) signals in the presence of the reagent. The operating principle is similar to microscopy-based live/dead staining, in which a membrane-impermeative DNA stain (typically, propidium iodide [PI]) is excluded from intact cells but penetrates and stains the DNA of membrane-compromised cells. In live/dead staining, inactivated cells are quantified relative to total cell counts by fluorescence microscopy or flow cytometry (11) rather than by PCR. The second strategy, termed “molecular viability testing” (MVT), correlates viability with the ability to rapidly synthesize a macromolecule (a species-specific rRNA precursor, or pre-rRNA) in response to a brief nutritional stimulus (12,–14). Pre-rRNA synthesis upon nutritional stimulation is detected by reverse transcriptase-qPCR (RT-qPCR) measurement of species-specific pre-rRNA molecules. Pre-rRNAs in inactivated cells, and free nucleic acids, do not increase in numbers upon nutritional stimulation and therefore are excluded.
- #13 sperm MTT viability assay as a new diagnostic test for the discriminationof live sperm from dead sperm. Formation ofMTT formazan granules or spikes around the spermmidpiece show that sperm mitochondria contains dehydrogenase,which is capable of converting MTT toMTT formazan, and the presence of MTT formazangranules in the midpiece region identifies that sperm as a live sperm.
- #17 Trypan Blue Trypan Blue is one of several stains recommended for use in dye exclusion procedures for viable cell counting. This method is based on the principle that live (viable) cells do not take up certain dyes, whereas dead (non-viable) cells do. Staining facilitates the visualization of cell morphology. NOTE: Trypan Blue has a greater affinity for serum proteins than for cellular protein. If the background is too dark, cells should be pelleted and resuspended in protein-free medium or salt solution prior to counting. Protocol for Viable Cell Counting using Trypan Blue Prepare a cell suspension in a balanced salt solution (e.g., Hanks' Balanced Salts [HBSS], Cat. No. H9269). Transfer 0.5 ml of 0.4% Trypan Blue solution (w/v) to a test tube. Add 0.3 ml of HBSS and 0.2 ml of the cell suspension (dilution factor = 5) and mix thoroughly. Allow to stand for 5 to 15 minutes. Note: If cells are exposed to Trypan Blue for extended periods of time, viable cells, as well as non-viable cells, may begin to take up dye. With the cover-slip in place, use a Pasteur pipette or other suitable device to transfer a small amount of Trypan Blue-cell suspension mixture to both chambers of the hemacytometer. Carefully touch the edge of the cover-slip with the pipette tip and allow each chamber to fill by capillary action. Do not overfill or underfill the chambers. Starting with chamber 1 of the hemacytometer, count all the cells in the 1 mm center square and four 1 mm corner squares (see Diagram I). Non-viable cells will stain blue. Keep a separate count of viable and non-viable cells. Note: Count cells on top and left touching middle line of the perimeter of each square. Do not count cells touching the middle line at bottom and right sides (see Diagram II). Repeat this procedure for chamber 2. Note: If greater than 10% of the cells appear clustered, repeat entire procedure making sure the cells are dispersed by vigorous pipetting in the original cell suspension as well as the Trypan Blue-cell suspension mixture. If less than 200 or greater than 500 cells (i.e., 20–50 cells/square) are observed in the 10 squares, repeat the procedure adjusting to an appropriate dilution factor. Withdraw a second sample and repeat count procedure to ensure accuracy. Calculations Cell Counts – Each square of the hemacytometer, with cover-slip in place, represents a total volume of 0.1 mm3 or 10-4 cm3. Since 1 cm3 is equivalent to approximately 1 ml, the subsequent cell concentration per ml (and the total number of cells) will be determined using the following calculations: Cells Per mL = the average count per square × dilution factor × 104 (count 10 squares) Example: If the average count per square is 45 cells × 5 × 104 = 2.25 × 106 cells/ml. Total Cells = cells per ml × the original volume of fluid from which cell sample was removed. Example: 2.25 × 106 (cells/ml) × 10 ml (original volume) = 2.25 × 107 total cells. Cell Viability (%) = total viable cells (unstained) ÷ total cells (stained and unstained) × 100. Example: If the average count per square of unstained (viable) cells is 37.5, the total viable cells = [37.5 × 5 × 104] viable cells/ml × 10 ml (original volume) = 1.875 × 107 viable cells. Cell viability (%) = 1.875 × 107 (viable cells) ÷ 2.25 × 107 (total cells) × 100 = 83% viability
- #18 Apoptosis is a complex processes involving a cascade mechanism that employs many proteins. However, the key enzymes in this process are the caspases, a family of cysteine proteases that control and mediate the apoptotic response. Virtually all animal cells contain caspases, but they occur as inactive zymogens that can do no harm. There are various triggers that can lead to their activation, which usually occurs through proteolytic processing of the zymogen at conserved aspartic acid residues. Needless to say, their activation and suicidal function is highly regulated. Once activated caspases act as cysteine proteases, using a cysteine side chain for catalysing peptide bond cleavage at aspartyl residues in their substrates. The name “caspase” denotes their function: Cysteine-dependentASPartyl-specific proteASE. There are many such caspases within an organism, which work together in a proteolytic cascade to activate themselves and one other. Cascades are effective means of amplifying a signal to give a much larger response than could be achieved through a single enzymatic reaction.
- #19 Easy, Colorimetric Test for Cell Death The easiest way to measure cell death is by measuring lactate dehydrogenase (LDH), a stable cytoplasmic enzyme which is present in all cells but only released when the plasma membrane is damaged. The LDH Cytotoxicity Detection Kit provides a simple and precise colorimetric assay for LDH activity: a two-step enzymatic reaction creates a formazan dye that is easily measured by A492. Why Use the LDH Cytotoxicity Detection Kit? Sensitive assay: Detect as few as 2,000 dead or damaged cells per well. Accurate results: The amount of formazan dye you measure is directly proportional to the number of dead or damaged cells. Cost-effective: Get your LDH assay for a better price. Fast, Simple LDH Assay Protocol The LDH assay does not require prelabeling or washing steps. The entire procedure can be performed in a single 96-well plate.