I am culturing SH-SY5Y cells in 96-well plates at a seeding density of $1.2 \times 10^5$ cells/mL. I differentiate the cells with retinoic acid for 6 days before giving them treatments. The cells look okay under microscope after the treatment. However, every time I read the plates, I get absorbance values of less than 0.2 (including my control).

I have tried increasing the volume of MTT reagent (5mg/mL), increasing the incubation time, but in vain.

Increasing the cell density indeed increase the absorbance, but the outcome seems illogical. A seeding density of $2 \times 10^6$ cells/mL gives absorbance values of 0.4+ (less than 0.5).

I am pipetting the mtt reagent and media out one-well-by-one-well using a micropipette. Assuming no pipetting errors and loss of cells, what are the possible causes of getting these low absorbance values?


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