2.4.1. MTT-viability test

To test the viability of cultured cells

1. Collect a sample of adherent cells which were incubated at minimum for 24 h at 33°C in a microtitre plate.

2. Centrifuge the plate for 10 min at 210 rcf (Eppendorf 5415 R, rotor A-2-DWP)

3. Aspirate the medium using a vacuum pump.

4. Cover the cells with 100 µl of freshly prepared BM3 medium supplemented with 250 µg/ml penicillin/streptomycin-solution (Roth, HP10.1) and 2.5 % antibiotic/antimycotic-solution (Sigma-Aldrich, A5955).

5. Incubate for 72 h at 33°C in a cooling incubator.

6. Centrifuge the microtitre plate for 10 min at 210xg to pellet the cells.

7. Aspirate the medium carefully without scratching the cells.

8. Add 100 µl of 0.5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT)-solution in BM3medium (Table 1) supplemented with 250 µg/ml penicillin/streptomycin-solution and 2.5 % antibiotic/antimycotic-solution.

9. Incubate at 33°C for minimum 3 h.

10. Centrifuge the plate again at 210 rcf for 10 min

11. Carefully aspirate the medium.

12. Add 100 µl of dimethylsulfoxid/acetic acid/sodium dodecyl sulphate (89.4 %/0.6 %/10 %) and incubate the plate for 5 min at room temperature on a shaker (Heydolph, Polymax 1040) for cell lysis

13. Analyse the viability-related colour in an ELISA reader (BioTek, Synergy HT) with 595 nm excitation wavelength.

Colorimetric intensity depends on individual cell type and doubling time. Percentage of viable cells should be between 80 and 100 % depending on cell strain.