Hi. I have a problem with MTT assay in my cell line. My cell line is B65 and I want to treat these cells with a plant extract. For density set up, I seeded cells in 96 well plate for 3 density: 103,104 and 105 cell/well. Then I treat cells with extract concentrations: 10-30-50-100 and 200 µg/ml (triplicate). In 103 density, control absorptions were: 0.412,0.348,0.342. For extract concentrations, absorptions were less than control and for 100 & 200 µg/ml, it reached 0.048, 0.05 etc.

In 104 density, control absorptions were: 0.612, 0.625 & 0.6. For extract concentrations of 10-30 & 50 µg/ml, absorptions were above control, but for 100 & 200 µg/ml, absorptions were0.048, 0.05 etc.

In 105 density, control absorptions and extract concentrations of 10-30 & 50 µg/ml w ere ≥1.

Control cells absorptions must be 1? My absorptions 0.3 or 0.6 are wrong?

My MTT protocol: after 24h of treatment, I add 20 µl MTT solution (MTT in PBS) into every well (in which there was 200 µl media+extract). After 3h, I remove 170 µl of media and add 190 µl DMSO. I put plate on shaker for 15 min and then I read with eliza reader in range: 460- 680 nm.

why sometimes I observe an suddenly incresed absorption in high concentration of extract? Can any one help me?

thanks