MTT assay problems (Cells just come off after incubation) - Cells just come off after incubation in 37 degree (Mar/25/2011 )

Hi, I am using a cancer cell line to test for a drug for cell proliferation rate.


The procedure i followed was,
1) plate the 96 wells with 10,000 cell/well, incubate for 24hrs
2) Add drug at different concentrations and incubate for 24hrs
3) add MTT in 500ug/ml and incubate for 4hrs
4) Centrifuge at 3800 rpm for 10 mins at room temp.
5) Remove supernatant and dissolve in DMSO
6) Read at 570nm


The cells come off in step (5)


Please Help ~~~

Is there a reason for removing supernatant? Typically it is preferred to add the solubilzation soution directly to the cells as the formazan crystals can be on and around the cells. Only sampling supernatant will likely cause unreliable results.

Also, if your intention is to use the cells for some other type of assay after the MTT measurement, I would warn against it. In our experience MTT can impact cell biology fairly substantially.

If you have any questions even if unrelated to any products, please don't hesitate to contact Promega Technical Services.

Regards,
Kevin