Hi guys. Im rescently having trouble with my MTT assay using RAW 264.7 CELLS.
that when ever i aspirate MTTreagent and spent media before adding DMSO, my crystals gets rid off from the bottom of 96 well plate.
Im wondering whether i can just leave 20 micro liters then dry the rest in 37'C incubator.
Here are my protocols.
I treated RAW(50% CONFLU) with my samples for 24 h
Then added MTT reagent for 2h (upon adding mtt reagent, confluency of negemative control = 80%)
Then centrifuged 2000 rpm for 5min then
Aspirated the supernatant and HERE IS THE PROBLEM whenever i tried to aspirate those spent media and MTT sup, i see some formazan crystals stuck on the end of pipette tip.
So...id like to ask whether is it alright to just leave about 20uL of MTT in the 96well prior to adding DMSO? OR should i perfectly dry then in the dark at what temperature?
thanks for thy help.
God bless.
Ah. And one more thing. Id like to ask is that
My readings of negative contol is around 3.5~4.0 (570nm)
I heard that OD below 1.5 is desirable.
So i diluted then 5x to get around 1.3 OD
when I calculated the viability,
The non_dilited OD data had shown that my samples treated cells are about 90% viable ocmpared to control.
However, when i used 5x diluted data, calculated results come out to be 70% viable.
Which data should i trust?
I would recommend extending your interest WST or others. For example:
Article Fibroblast cell lines from young adult mice of long-lived mu...
Donghan Kim
While performing MTT assay it is highly unlikely to get crystals on your pippet tip. Unless you don't add MTT dye slowly on the walls.
In your case RAW 264.7 cell line which is semi-adherent in nature, So in my experience it is not advised to aspirate the MTT media solution. You can add directly 50% DMF & 20% SDS after your MTT treatment. DMF will dissolve formazan crystals and SDS will lyse the cell wall. Whereas keeping 20ul of residual media will dilute DMSO and wont give better lysis.
There are many different MTT assay protocols.
Instead of 2000 rpm, you could try 4000 rpm. I use this, but sometimes I do have problems with crystals getting suspended.
Avoid putting the tip too deep. Try to lean the plate and aspirate from the wall.
About OD. How much DMSO do you use to dissolve the crystals? I use 150 uL, but you can find a range of 100-200uL if you look for some other protocols.
It is desireble to have OD below 1.5 due to loss of sensitivity.
I would not recommend that you leave it to dry in the incubator. Residual enzymes and cells could reduce more MTT.
Look for more protocols, compare them and see which fits best.
Thank you for your answers, it helped me a lot.
I just have found best protocol using DMSO as solubilizer.
I leaed plates and aspirated almost all media for the well, leaving only 10uL, (I couldnt aspirate all of them because i was afraid of scraping crystals at the bottom. then I used 200 uL DMSO and shaked for 20min for solubilization and measured OD at 570nm and the results of triplicates were so consistent.
I'd like to forward this protocol to anybody who are using DMSO in MTT.
Best regards.
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