April 14, 2009
MTT assay
Step
Activity
Cell seeding
1.
Select the cell culture at approximately 80% confluence (logarithmically growing cells)
2.
Harvest the cells by trypsinization (0.25%; 2 min)
3.
Prepare the cell suspension (~5,000 cell/well -- 5x104 cells/ml)
4.
Intr
oduce 100 l aliquot of the cell suspension into each well of the test plate (keep at least
3 well empty for the blank)
5.
Culture the cells in a CO2 incubator (overnight/ 2 nights, it depends on the cell type)
Treatment
6.
After incubation, discard the culture medium by inverting the plate (always discard the
liquid by inverting the plate, then invert the plate on paper towels to absorb the remaining
drops of liquid).
7.
Add each well with 100 l PBS, then discard the PBS.
8.
Add 100 l of the test chemical dissolved in culture medium into each well (do in
triplicate) and then culture for 24/48 hours.
MTT Staining
9.
Discard the culture medium containing the test chemical.
10.
Wash with 100 l PBS.
11.
Add 100 l of medium containing MTT at the concentration 0f 0.5 mg/ml. Incubate the
plate for 3-4 hours in CO2 incubator.
12.
After incubation, discard the medium containing MTT.
13.
Wash with 100 l of PBS.
14.
Add 200 l of acid-isopropanol (1 portion of 4N HCl : 100 portion of isopropanol)
Note: another procedure to stop the MTT reaction is to add 100 l of 10% SDS in 0.01 N
HCl directly into the medium containing MTT and incubate overnight in the dark. Shake
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10 min before measuring the OD590.
15.
Agitate the plate by microplate shaker for 10 min to extract and solubilize the formazan.
16.
Measure the OD590 by automatic microplate reader.
Calculation of Cell Viability and IC50
17.
Determine the cell viability by the formula
Cell viability (%) = (OD value of treated well – OD value of the blank) x 100%
(OD value of untreated well – OD value of the blank)
18.
Determine IC50 value by probit / logit analysis by SPSS or linear regression by Microsoft
Excell.
Note:
1. To make 10 ml of MTT stock solution: measure 50 mg MTT and dissolved in 10 ml PBS.
2. To make 10 ml of MTT working solution: add 9 ml culture medium into 1 ml MTT stock
solution.
3. To make 20 ml acid-isopropanol: add 20 ml isopropanol into 200 l 4 N HCl.
4. To make 10 ml of 4N HCl: add water into 2.5 ml 12 N HCl (37% -- stock) to make final
volume of 10 ml.
5. To make 10% SDS in 0.01 N HCl: dissolve 10 g SDS in 100 ml of 0.01 N HCl.
6. To Make 0.01 N HCl: dissolve 1 ml of 1 N HCl in 100 ml water.
For WiDr cells: seed 5000 cells/well, incubate 24h prior to treatment.
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