MTT assay:
The MTT assay is a colorimetric assay used to assess cell metabolic activity. It involves the
reduction of the yellow tetrazolium salt (MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide) to purple formazan crystals by metabolically active cells. MTT is membrane-permeable
and is especially suitable for adherent cell cultures.
MTT Medium Preparation for 1 Well (24-Well Plate)
Component
Volume for 1 Well (12-Well Volume for X Wells
Plate)
MTT Solution (5 mg MTT/mL 20 µL
PBS)
Cell Culture Medium
200 µL
1. Prepare MTT Medium
Mix the MTT solution with the cell culture medium in the specified ratio.
2. Wash Cells
Rinse the cells with PBS to remove any residual medium.
3. Add MTT Medium to the Wells
Pipette the prepared MTT medium into each well.
4. Incubate
Place the cell plates back into the incubator (37°C, 5% CO₂ ) and incubate for 40 minutes.
5. Remove MTT Medium
After incubation, aspirate the MTT medium (220 µL) from each well.
6. Add Isopropanol
Add 400 µL of isopropanol to each well to dissolve the formazan crystals.
7. Incubate on Thermo-Shaker
Incubate the plate for 10 minutes on a thermo-shaker at 22°C and 300 rpm (e.g., in the
ELISA lab).
8. Transfer Samples to a 96-Well Plate
Transfer 100 µL of the solution from each well into a 96-well microtiter plate in duplicates.
Add 1 well for blank (only isopropanol).
9. Measure Absorbance
Measure the absorbance at 590 nm using a plate reader (e.g., Tecan plate reader). The
template for analysis is saved in the A. Festag folder.