Precipitate forming after MTT ! - I cant correctly read my assay, help please (Mar/04/2011 )

Hello all,

Im having some trouble with MTT assays and couldnt find any info regarding this....

I have done several MTT assays in the past with cervical cancer cells, and never had a problem... I always used isopropanol to dissolve the formazan crystals, but, recently, I have been working with brain tumors cells and neuroblastoma cells, and, when I add the isopropanol, the crystals disolve but the liquid become opaque.... If I read the plate, the numbers are not accurate as Im reading absorbance... If I leave the plate there overnight, I can see a white precipitate at the bottom of the wells...

My question is... Have anybody else here experienced this white precipitate ?? What should I use to dissolve the formazan ??

Could it be the myelin in the cells... or... ideas !?

Thank you so much !!!

Have you tried using DMSO instead of isopropanol?

What is the pH of the isopropanol you are using?

Oh.... I have no idea !! I will check on monday.... should pH be 7.5 or... ??

I think I will try DMSO at least in one well next week... We just have DMSO for freezing cells you know...

Could somebody please plesae help me ??

I then tried the SDS/HCl solution and it never turned purple !!! just yellow !! What is happening in my MTT !?

The media for your cells - what % FBS is it (assuming it is supplemented with some amount of FBS)?

When you say "I then tried the SDS/HCl solution and it never turned purple !!! just yellow !!" do you mean that after the MTT solution was removed, and the cells/remaining dye were resolubilized in the isopropanol or SDS solution, the solution turned yellow? Or are you referring to the yellow color of the solution of MTT added to the cells prior to the solubilization in DMSO/isopropanol/SDS?

How long are you incubating the MTT dye in with the cells prior to attempting to solubilze it in SDS/etc?