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MTT assay problem! plz help!


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#1 akrivi

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Posted 10 February 2009 - 06:23 PM

hello! I have been doing MTT assays in 96 well plates with no problems but i have recently swiched to 24well plates and my normally adherent cells now detach from the well. that way i can't aspirate the mtt and add DMSO to solubulize the crystalls. I use 5mg/ml mtt(sigma) in pbs with sodium succinate. I add 100ul mtt in a 24-well with 700ul media and after 3 hrs i aspirate the media+ mtt and add 300ul DMSO.

If someone could help me plz cause i'm wasting my precious cells!

#2 cotchy

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Posted 11 February 2009 - 05:11 AM

hello! I have been doing MTT assays in 96 well plates with no problems but i have recently swiched to 24well plates and my normally adherent cells now detach from the well. that way i can't aspirate the mtt and add DMSO to solubulize the crystalls. I use 5mg/ml mtt(sigma) in pbs with sodium succinate. I add 100ul mtt in a 24-well with 700ul media and after 3 hrs i aspirate the media+ mtt and add 300ul DMSO.

If someone could help me plz cause i'm wasting my precious cells!


I have done the MTT assay many times but have never used a 24 well plate for it, is there a reason you have to use a 24 well plate?

The reason for detachment could be that the plates are from different manufacturers and the 96 well plates are made of one plastic which allows cells to attch easily and maybe were also coated in collagen or something similar, and the 24 well plates are not, have you looked into this?

One tip we were often told (although i never did use it) was instead of aspirating was to just turn over the open plate onto tissue spayed with 70% ethanol to remove the liqiud and can be done after washing and adding DMSO aswell.

This removes the aspiration step, but as i said i never used it because it does remove some of the cells from the middle of the wells but not that many.

You could give it a try and see is it any better, but first i would compare the plastic/coatings on your 24/96 well plates

#3 little mouse

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Posted 11 February 2009 - 05:31 AM

you could centrifuge the plate before to remove the supernatant.
we do that for cells growing in suspension.

#4 akrivi

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Posted 14 February 2009 - 06:45 PM

hello! I have been doing MTT assays in 96 well plates with no problems but i have recently swiched to 24well plates and my normally adherent cells now detach from the well. that way i can't aspirate the mtt and add DMSO to solubulize the crystalls. I use 5mg/ml mtt(sigma) in pbs with sodium succinate. I add 100ul mtt in a 24-well with 700ul media and after 3 hrs i aspirate the media+ mtt and add 300ul DMSO.

If someone could help me plz cause i'm wasting my precious cells!


I have done the MTT assay many times but have never used a 24 well plate for it, is there a reason you have to use a 24 well plate?

The reason for detachment could be that the plates are from different manufacturers and the 96 well plates are made of one plastic which allows cells to attch easily and maybe were also coated in collagen or something similar, and the 24 well plates are not, have you looked into this?

One tip we were often told (although i never did use it) was instead of aspirating was to just turn over the open plate onto tissue spayed with 70% ethanol to remove the liqiud and can be done after washing and adding DMSO aswell.

This removes the aspiration step, but as i said i never used it because it does remove some of the cells from the middle of the wells but not that many.

You could give it a try and see is it any better, but first i would compare the plastic/coatings on your 24/96 well plates

Thank you very much.It was the coating after all. I ordered new plates and I had no problem!




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