Cell viability assay

cell viability assay

1. MTT ASSAY

2. INTRODUCTION 2

3. MTT 3-(4,5-di M ethy T hiazol- 2-yl)-2,5- diphenyl T etrazolium bromide 3 Dimethyl thiazolyl Diphenyl tetrazole

4. MTT , a yellow  tetrazole is reduced to  purpleformazan  in living cells.   A solubilization solution   dimethyl sulfoxide an acidified ethanol solution Isopropanol detergent sodium dodecyl sulfate in diluted hydrochloric acid) is added to dissolve the insoluble purple  formazan  product into a colored solution. 4

5. PRINCIPLE 5

6. PRINCIPLE The  MTT assay  is a colorimetric assay for assessing cell metabolic activity.  NAD(P)H-dependent cellular oxido reductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble  formazan , which has a purple color.  6

7. MTT ASSAY: Principle : This colorimetric assay uses reduction of a yellow tetrazolium salt (3-(4,5- di methyl thiazol -2-yl)-2,di phenyl tetrazolium bromide, or MTT) to measure cellular metabolic activity as a proxy for cell viability. 7

8. MTT assays are usually done in the dark since the MTT reagent is sensitive to light. 8

9. Principle Viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT reagent to formazan , an insoluble crystalline product with a deep purple color . Formazan crystals are then dissolved using a solubilizing solution ( organic solvent s- eg . Isopropanol /DMSO) and absorbance is measured at 500-600 nanometers using a plate-reader. 9

10. The darker the solution, the greater the number of viable, metabolically active cells The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer. The degree of light absorption depends on the solvent. 10

11. Other than MTT XTT XTT  (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) has been proposed to replace MTT, yielding higher sensitivity and a higher dynamic range. The formed formazan dye is water-soluble, avoiding a final solubilization step 11

12. MTS   MTS  (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), in the presence of phenazine methosulfate ( PMS ), produces a formazan product that has an absorbance maximum at 490 nm in phosphate-buffered saline. The MTS assay is often described as a 'one-step' MTT assay. 12

13. WST WST-1 and in particular WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium), are advantageous over MTT in that they are reduced outside cells, combined with PMS electron mediator, and yield a water-soluble formazan . Finally, WST assays (1) can be read directly (unlike MTT that needs a solubilization step), (2) give a more effective signal than MTT, and (3) decrease toxicity to cells (unlike cell-permeable MTT, and its insoluble formazan that accumulate inside cells) 13

14. Tetrazolium dye assays can also be used to measure Cytotoxicity (loss of viable cells) Cytostatic activity (shift from proliferation to quiescence [ it is a state of quietness or inactivity) of potential medicinal agents Cytostatic activity of toxic materials.  Number of viable cells present 14

15. CELL VIABILITY ASSAY

16. 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, – nucleotide uptake activity 16

17. 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). 17

18. 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 18

19. Procedure 19

20. 20

21. MTT ASSAY PROTOCOL: Trypsinization or scrapping the cells Resuspend or splitting cells at 96 well plate Check the cell confluency (if 90%-100% of the cell then it is ready for using) 100~ 200 µl of desired drugs or compound in each well (usually 1 compound /drugs should have triplicates) Incubate the cells (12~24 hours) Dissolve 1mg MTT in 1ml PBS solution then the molar concentration will be 2.41m M 21

22. In each well add 20 µl MTT reagent and rest 160 µl culture media and incubate it for 3~4 hours check the cells to see the purple precipitation and the purple color is clearly visible then discard the media carefully as it does not drain the cells. after discarding media add 100 µl DMSO/ isopropanol cover it with aluminum foil and mildly shake it for 10 ~15 minutes Read plate in fluorescent Reader – measure OD in 570nm (background wavelength is 630nm). 22

23. MASS = CONCENTRATION X VOLUME X MOLECULAR WEIGHT Interpreting results The absorbance reading of the blank must be subtracted from all samples. Absorbance readings from test samples must then be divided by those of the control and multiplied by 100 to give percentage cell viability or proliferation (see formula below). Absorbance values greater than the control indicate cell proliferation, while lower values suggest cell death or inhibition of proliferation. 23

24. % Viable cells = abs ( sam )- abs (blank) abs (control) – abs (blank) TROUBLE SHOOTING :REAGENTS: MTT is light-sensitive, and should be kept in the dark as much as possible. It is always a good idea to cover MTT bottles with foil . Make sure you do not contaminate your MTT stock. If it is green or blue (it should be yellow), you can assume it has either been contaminated by cells or bacteria, or exposed to light. If properly looked after and stored at 4°C, your MTT stock can last up to 18 months. Always handle MTT using the appropriate personal protection equipment, as it is can cause skin and eye irritation. To dissolve formazan crystals , you can use DMSO, acidified isopropanol , or SDS. Test to find out which works best for your experiment. 24

25. Absorbance values Your absorbance readings should fall between 0.75 and 1.25. If your readings are too low, try increasing the number of cells plated or the incubation time, and make sure your cell culture conditions are appropriate. On the other hand, plating too many cells per well will yield very high absorbance readings, as will contamination by yeast or bacteria. To determine the optimal cell number per well, prepare and plate serial dilutions of cells in medium from 10 6  to 10³ cells/ mL. This will help you decide which number of cells yields appropriate absorbance values. Always include a positive control (untreated cells) and a blank (well containing medium only), and plate these and samples in triplicate. Always wash cells with PBS (step 2) before adding MTT in order to remove dead cells and cellular debris, which could give inaccurate results. 25

26. ADVANTAGES : No transfer of the cells, the entire assay is performed in a single microplate MTT is metabolised by all cells , the assay can be used with all cell types. In expensive. DISADVANTAGES: Cannot take multiple time points in a single assay. Cells with low metabolic activity ( eg : lymphocytes) must be used in high numbers. 26

27. APPLICATIONS: Cell proliferation assays Cytotoxicity analysis Apoptosis screening Anticancer drug Predictive drug testing for tumors Genotoxicity 27

28. REFERNCE: MTT assay protocol by Masuma akter https://blog.quartzy.com/2017/05/01/cell-viability-assays-mtt-protocol 28