17. Bioprotocols--MTT2

MTT Assay for Cell Proliferation

MTT stock solution:

5 mg/ml MTT (Promega) in PBS (pH 7.4).

This solution is filtered through a 0.2 mm filter and stored at 4 centigrade.

MTT working solution:

1:10 dilution of the 5 mg/ml MTT stock in non-serum cell culture medium.

Procedure:

1. Plate cells into a plate as usual. (Note: we need some blank wells without cells for control)

2. Wash each well with PBS.

3. Prepare MTT working solution.

4. Add MTT working solution into wells being assayed, (for example 1.0 ml for each well of 12-well plate, 100 米l for each well of 96-well plate). Incubate at 37 centigrade CO₂incubator for 30 min to 2 hours (this time depends on cell density and cell type).

5. At the end of the incubation period, the medium is removed.

6. The converted formazan is dissolved with the same amount of DMSO as used for MTT. Shake the plate gently for 2-3 min to make sure the converted formazan dissolves completely.

7. Absorbance of the color is measured at a wavelength of 550 nm with a microreader.

MTS Assay for Cell Proliferation

It just works the same way as MTT Assay, except:

1) The formed formazan can dissolve with water, so it's not necessary to use DMSA as solvent.

2) The working can be added directly to the medium without replaced or wash it, but we suggest

the medium be removed and the wells be washed with PBS to get better results.

3) The OD value should be read at 490nm wavelength, not 550nm

4) The MTS working solution should be mixed with MTS and buffer according to the manufacturer's instruction.