Just wonder if anybody can help me out.
I am using MTT Cell Proliferation Assay for cell growth and cytotoxicity assay. During optimization assay, I am getting quite low absorbance value i.e around 0.3-0.4 for very high cell concentration i.e 10^5 cells/mL.
1. The reaction of MTT with my blank (i.e media only) is quite strong. I am using Leibovitz media supplemented with 10% FBS. The absorbance is around 0.7-0.8. I verified with another media, RPMI 1640 and the absorbance is much higher than Leibovitz. Around 2.0. Due to this I am wondering if I should take out the media before putting the MTT. Do u think this is OK with the assay and will give better absorbance value?
2. After putting MTT, I kept the plates in the incubator for 4 hours. However the formazan formattion was very weak. When I extended to 24 hours it was better, and absorbance reading was higher. Is there any adverse effect eg toxicity to the cells due to the prolonged exposure to MTT?
3. Referring to ATCC determining optimal cell count protocol, the ideal number of cells to be used should yield an absorbance of 0.75-1.25. Do you know why is that figure? Does this apply to all cell lines? I am using colorectal cancer cell lines SW620 and SW480. Will also use few other cell lines i.e SW1116, SW948, CCD-18-Co after this. So far, from my assay, the absorbance values for 3000-7000 cells/well are quite low, around 0.3-0.4.
4. How do you find the optimum wavelength for your assay? I use 550/690nm bcoz it was the recommended wavelength from the brochure. But just wondering whether my low absorbance is due to the wavelength.
Many many thanks ahead for your help.