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Viablity assay

am birhanu, from universty of Gondar

  • Sé el primero en comentar

Viablity assay

  1. 1. DEPARTMENT OF IMMUNOLOGYAND MOLECULAR 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. 2. Objective At the end of 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. 3. Introduction To Viability Assay 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. 4. Features of viable cell • 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. 5. Introduction To Viability Assay 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. 6. Application of viability assay • 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. 7. Classification of viability assays 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. 8. Common methods used in viablity assay MTT cell viability assay WST methods Trypan Blue Lactate dehydrogenase (LDH) Flow cytometry PCR ??????????? 5/29/2017 viablity assay 8
  9. 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. 10. Principles of MTT viablity assay 5/29/2017 viablity assay 10
  11. 11. Procedure for MTT assay 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. 12. Sperm MTT assay 5/29/2017 viablity assay 12
  13. 13. water-soluble tetrazolium salt colorimetric 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. 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. 16. Trypan Blue • widely used 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. 17. Cell viablity, appoptosis and necrosis assay 5/29/2017 viablity assay 17
  18. 18. 5/29/2017 viablity assay 18
  19. 19. ACKNOWLEGMENT I would like to 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. 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. 21. 5/29/2017 viablity assay 21 Thank u for Your Attention

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