I have been doing MTT assay for testing cytotoxicity of thio-containing compound. After adding 50 microlitter/well, I put the 96-well plate back to incubator within 4 hours. Instead of appearing the purple after dissolving formazan cystal by DMSO, there were some deep purple dots appearing randomly (I enclosed the picture) , the colorimeter also shown me very high intensity. Are there any posibilities related to contaminations to exclucidate this problems?
Thank you so much for any advice.
When stored for long time period MTT can soak humidity from air. And that leads to formation of insoluble crystals of MTT. Then such a crystal can randomly be placed into wells, thus some wells have higher concentration of MTT. Check your MTT powder if there are lumps, as well the MTT solution if there are any speckles.
Agnieszka Bojko Thank for your response.
When I prepare MTT solution, I aslo use millipore membrance 0.22 micrometer to filter it before diluting to get 5mg/mL; therefore I'm convinced that MTT solution is uniform.
Anyway I'm gonna double check my MTT powder.
Nguyen Thi Thu Suong No problem! But if you are filtering your MTT solution there should be no lumps. Maybe the other reason for that are cells that are clumped, thus forming clusters. Maybe look under the scope to see whether this is the case. If so, try to pipette those wells and check wether it dissolved. Sometimes it happens.
Hi Nguyen Thi Thu Suong ,
Yes, that is may be a contamination,
Before adding the MTT solution to the well, check the all 96 well under microscopy. If the well is good, check MTT solution sterility.
Agnieszka Bojko Prasath Manogaran Thank for offering me these possibilities, I've just checked Hela Cells under microscope and it 's definitely got clusters even though I pipetted cell suspension so many times. It seems like the next trouble is coming up ^^
Thank you everyone again
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