Hi everyone,
I am trying to do an MTT with caco-2 cells. I am following the same general protocol for an MTT for a 24 well plate.
1. Seed cells 48h
2. Add 100ul of MTT solution
3. Incubate for 3h at 37 degrees 5 % CO2
4. Let formazan crystals dry
5. Add 200ul of DMSO and dissolve crystals
6. Read on plate reader at 570nm reference wavelength 620
I am getting low absorbance values for this 0.3 - 0.6. Am I missing anything to get such low values? Do I need to do the experiment on differentiated or undifferentiated cells?
Hello Samanvita Ravikanti
The fourth step is incorrect (let formazan crystals dry).
Avoid drying of the formazan crystals. When formazan crystals dry, they will become difficult to dissolve leading to incomplete solubilization and inaccurate measurements, potentially causing underestimation of cell viability.
Instead, add the solubilization solution (like DMSO) immediately after the incubation period with MTT, while the formazan crystals are still wet and fresh. You should gently shake the plate after adding DMSO to ensure complete mixing and proper dissolution of the crystals.
You remove the medium containing MTT after incubating for 3h at 37 degrees 5 % CO2, and add DMSO while the formazan crystals are still wet. Prompt solubilization is necessary to obtain meaningful data from the assay.
Best,
I agree with Malcolm. Remove media with MTT solution and immediately add DMSO to solubilize.
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