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Adherent cells washing off in 96 well -MTT


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#1 chopramo

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Posted 08 August 2010 - 10:46 AM

Hi all

I am using INS-1 cells and treating with a particular drug and measuring cell viability using MTT assay (96-well plate). When I remove the media to add the MTT, the cells just come off and obviously the results are no longer consistent. I wanted to know if I can do something so the cells wont come off, like using something to get the cells to stick. Also, I tried the neutral red assay which requires me to wash the excess neutral red solution with PBS (x2) and I cannot do that because my cells will just wash off and I will be left with empty wells.

I use 20K cells per well, I have tried seeding them for 12, 24, 48 and 72 hours before using them and I have this problem every time. Yes, I can turn to other techniques but MTT and Neutral red assay are very good for preliminary investigations.

Please help!!!

#2 SuMi

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Posted 15 August 2010 - 07:30 AM

Could you spin the plate before you remove the supernatant and in between PBS washes?

#3 chopramo

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Posted 15 August 2010 - 11:57 AM

Could you spin the plate before you remove the supernatant and in between PBS washes?

Thanks for the reply, I will try and hope this works...
cheers

#4 gilahari

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Posted 15 August 2010 - 12:05 PM

switch to the atp viability assay (Cell titer glo from promega). no need to change media or wash cells. just add the reagent and read luminescence. consistent results.

if you have to use MTT then what worked for me was to not remove all the media from the 96 well plate. leave a few uL behind. It is a pain but you lose much less cells if you are gentle enough.

#5 chopramo

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Posted 15 August 2010 - 01:52 PM

switch to the atp viability assay (Cell titer glo from promega). no need to change media or wash cells. just add the reagent and read luminescence. consistent results.

if you have to use MTT then what worked for me was to not remove all the media from the 96 well plate. leave a few uL behind. It is a pain but you lose much less cells if you are gentle enough.


Well I have tried to use other assays, they are fine but for my project I need to use cell viability assays a lot and MTT is a cheaper option (if it works well).

I will try to follow your advice. I have also tried WST-1 where I do not require the removal of media but the issue is that WST-1 reacts with the reagents I am testing.

Anyways thanks for the advice

Cheers




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