HT-29 colon cancer cells were incubated with cell-free supernatants of Lactobacillus spp. for 48 & 72 hours.
When observed under the microscope, control cells showed growth over 48 to 72h and appeared confluent. Moreover, purple formazan crystal formation was greater in 72h control compared to 48h control (see attached pictures) confirming cell proliferation and active cell metabolism.
However, when 72h control cell crystals were dissolved in isopropanol, their absorbance was lower than expected (OD 2.57 vs OD 3.0) and almost in the level of 48h control cells (OD 2.25). Moreover, the low concentration Lactobacillus-treated cells were actually those who yielded the expected OD of 72h control cells (OD 3.4).
Reproducibility was quite good, so maybe we are looking for systematic rather than random errors.
Any ideas? Please feel free to suggest.
Dear Christos Velesiotis
First, the OD values you shared are too high. Ideally you should try to adjust OD of your non-treated samples to 0.7-1.2 range for an MTT assay. You may increase the volume of isopropanol but I think you might just have too many cells to begin with. Therefore another strategy would be to decrease the number of cells you seeded. I know this does not answer your question exactly but at high absorbance levels, you cannot compare two values with confidence.
Second, the cells may go through growth arrest via contact inhibition, if cell density is too high. It may even induce apoptosis. This may explain having lower OD for 72h non-treated cells. Both problems can be avoided by seeding fewer cells.
Cheers
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