Viability testing of cells

techniques to test the viability of cells

1. VIABILITY TESTING OF CELLS Presented by, D.Irene . 16PGZ001.

2. viability The capacity of the cells to be alive, capable of living and reproducing. Viability of the cells represents the capability of their existence, survival and development.

3. Cell viability assays  assess how healthy the cells are by measuring markers of cellular activity. Cell viability determines how well or how poorly cells will respond to stress stimuli   proliferation assays  are used to monitor the growth rate of a cell population or to detect daughter cells in a growing population.  Cytotoxicity assays  are used to determine the number of live and dead cells in a population after treatment with a drug Apoptosis assays  look at how cells are dying by measuring markers that are activated upon cell death. These are specific biochemical and morphological markers that do not occur in necrosis. apoptosis assays are used when we want to examine how pharmacological compounds kill cells; or why some cells/tissues do not respond to toxic compounds.

4. Why? Cell-based assays are often used for screening collections of compounds to determine if the test molecules have effects on cell proliferation or show direct cytotoxic effects that eventually lead to cell death.  Regardless of the type of cell-based assay being used, it is important to know how many viable cells are remaining at the end of the experiment. only a healthy culture will show reliable and reproducible results. 

5. How?? Example: Live cell count: 2,337,500 cells/mL ​Dead cell count: 50,000 cells/mL 2,337,500 + 50,000 = 2,387,500 cells 2,337,500 ÷2,387,500 = 97.9% viability

6. Viability assays are based on luminescence- based tests. radioisotope incorporation cellular respiration measurement of membrane integrity, 

7. Based on membrane integrity  The damage to membrane may occur due to cell disaggregation, cell separation or freezing and thawing. Membrane integrity can be determined by uptake of dyes to which viable cells are impermeable (e.g. naphthalene black, trypan blue, erythrosin )   Dye exclusion assay Dye uptake assay Enzyme release assays Labelled chromium uptake assay

8. DYE EXCLUSION ASSAY viable cells are impermeable to several dyes such as naphthalene black, trypan blue, eosin Y, nigrosin green and erythrocin B. (in plants- evan’s blue stain) The technique basically consists of mixing the cells in suspension with the dye observe under the microscopy. The stained cells and the total number of cells are counted. The percentage of unstained cells represents the viable cells. The major limitation of this assay is that reproductively dead cells do not take up the dye, and will be counted as though they are viable.

9. Dye uptake assay The viable cells can take up the dye diacetyl fluorescein and hydrolyse it to fluorescein . The latter is held up by the viable cells, as it is impermeable to membrane. The viable cells therefore emit fluorescein green while the dead cells do not. Thus, the viable cells can be identified.

10. Labelled chromium uptake assay Labelled chromium ( 51 Cr) binds to the intracellular proteins through basic amino acids. When the cell membrane is damaged the labelled proteins leak out of the cell degree of leakage is proportional to the amount of damage.

11. ENZYME RELEASE ASSAY The membrane integrity of cells can also be assessed by estimating the enzymes released. Lactate dehydrogenase (LDH) has been the most widely used enzyme for this purpose Principle : When using an LDH colorimetric assay, the amount of LDH released in the surrounding environment is measured with an enzymatic reaction which converts iodonitrotetrazolium or INT (a tetrazolium salt) into a red colour formazan . When the cell membrane is damaged lactate dehydrogenase (LDH), is released into the surrounding extracellular space. Since this only happens when cell membrane integrity is compromised, the presence of this enzyme in the culture medium can be used as a cell death marker. When LDH is present in the cell culture, it reduces NAD +  to NADH and H +   the catalyst ( diaphorase ) then transfers H/H +  from NADH + H +  to the tetrazolium salt INT forms the red coloured formazan salt. The amount of colour produced is measured at 490nm and is proportional to the amount of damaged cells in the culture.

12. BASED ON CELLULAR RESPIRATION Respiration of the cells measured by oxygen utilization or carbon dioxide production can be used to assess cell viability. This is usually done by using Warburg manometer.

13. Based on radioisotope incorporation By using radiolabelled substrates or metabolites, the radiolabel in the products formed can be detected. This method is particularly useful for the cytotoxicity assays of drugs Labelled nucleotides - Incorporation of ( 3 H) thymidine into DNA and ( 3 H) uridine into RNA are widely used for the measurement of drug toxicity. Labelled phosphate- The cells are pre-labelled with  32 P. When the damage occurs to cells they release labelled phosphate which can be measured. The efficacy of drugs can be evaluated by this approach.

14. Based on luminescence test The viability of cells can be measured with good sensitivity by estimating ATP levels by luminescence based test. The principle is based on the following reaction.

15. Tetrazolium Reduction Assays -MTT ASSAY The tetrazolium reduction assays measure some aspect of general metabolism or an enzymatic activity as a marker of viable cells. All of these assays require incubation of a reagent with a population of viable cells to convert a substrate to a coloured or fluorescent product that can be detected with a plate reader. MTT which is positively charged and readily penetrates viable eukaryotic cells 

16. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) substrate is prepared in a physiologically balanced solution, added to cells in culture, usually at a final concentration of 0.2 - 0.5mg/ml incubated for 1 to 4 hours The quantity of formazan (presumably directly proportional to the number of viable cells) is measured by absorbance at 570 nm using a plate reading spectrophotometer Principle : Viable cells with active metabolism convert MTT into a purple colored formazan product with an absorbance maximum near 570 nm . When cells die, they lose the ability to convert MTT into formazan, thus colour formation serves as a useful and convenient marker of only the viable cells. Note : The formazan product of the MTT tetrazolium accumulates as an insoluble precipitate inside cells as well as being deposited near the cell surface and in the culture medium. The formazan must be solubilized prior to recording absorbance readings. Various solubilization methods include using: acidified isopropanol, DMSO, dimethylformamide, SDS,

17. Other viability assays ATP test Calcein AM Clonogenic assay Ethidium homodimer assay Evans blue Flow cytometry Green fluorescent protein Methyl violet Propidium iodide, DNA stain that can differentiate necrotic, apoptotic and normal cells. Resazurin TUNEL assay

18. conclusion VIABILITY testing is of high importance in many areas of cell research including cytotoxicity tests based on cell and tissue cultures , the selection of proper tissue scaffolds for regenerative medicine ,quality assurance of products for transplantation research for cancer treatment and also for the inspection of hybridoma cells For the evaluation of cell viability, flow cytometry and microscopy are used besides the detection of cellular secretion products

19. References http://www.biology-online.org/dictionary/Viability http://www.abcam.com/kits/a-guide-to-cell-viability-proliferation-and-apoptosis-assays Ian Freshney , Glynn Stacy, Jonathan., Culture of human stem cells.,John Wiley publications.,2007.,pg-6. http://www.biologydiscussion.com/cell/cytotoxicity/cell-viability-and-cytotoxicity-5-assays/10557. A. Kummrow , M. Frankowski , N. Bock, C. Werner, T. Dziekan , J. Neukammer ., Quantitative Assessment of Cell Viability Based on Flow Cytometry and Microscopy., Cytometry Part A 83A: 197204, 2013. http://www.abcam.com/kits/a-guide-to-cell-viability-proliferation-and-apoptosis-assays https://info.gbiosciences.com/blog/why-is-lactate-dehydrogenase-ldh-release-a-good-measure-for-cytotoxicity