The control sample set of the MTT assay is shown to have detached from the plate as seen by the clear area (without the formazan crystals), when even cells that were treated with low levels of drugs show significantly less detachment.
Simple Protocol overview ( INS1 cells)
1) seed cells 5 x 10^6 cells/ml in 12 well plate (1ml each well)
2) after cells show normal morphology ( after around 30 hours), suction and change with drug mixed complete media
3) after 24 hour incubation
4) add MTT *
5) suction and insert DMSO to dissolve formazan and then detect with spectrometer
*I even modified the original step 4 which was actually suction then fill with MTT mixed complete media, because I thought the suction and exposure to air of the post-drug treated cells would be sensitive and hence led to the detachment.
Could anyone suggest where I would have allowed the control cells to have been exposed to stress that it would have led to the detachment?
For some reason the clear areas are always at the bottom of the well, forming a smile-like shape.
For reference: I suction from the top of the wells
Also, the control on the left picture was treated and handled almost identically to the 0.2uM sample on the picture on the right, but the drug treated sample seem to be in a better state than the control, which is very counter-intuitive since the drug does have some toxicity
A potetianlanswer:
If your drug treatment impairs cells growth/proliferation, the cells in the drug treated wells may have exhibited less growth and not reached confluence.
However if the DMSO control cells continued to grow as normal they may have reached confluence and begun to detach; many cell lines when they are "too dense" will start to detach; to rule this out I would recommend you decrease your cell density when you initially plate and see if this still occurs
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