MTT with DMSO or MTT with 10% SDS? - (Jan/15/2013 )

Hi all

Mine is adherent cell line..RIN cell line
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I have to optimise doses for alloxan and streptozotocin!

Recently

I opted out two methods after mtt asaay!!

One with DMSO...ie, carefully removing supernatant and adding 100mic..DMSO,..taking immediate reading and 20sec shaking!!
Other incubate overnight with 10% SDS(37C) ie 18 hour incubation... and later reading at 570nm

RESULTS

With 10% SDS results were good but triplicate values are not good enough
With DMSO results are varying....30% good result


QUERY

1)FOR ADHERENT CELLS WHICH SUITS BEST ??? DMSO OR 10% SDS?

2) IF DMSO? DO WE GOT TO TAKE IMMEDIATE READING OR 1 HOUR INC??
IF 10% SDS...ANY SUGGESTIONS???

Since SDS take18 hour incubation i want to go with DMSO...for reading!!

Please suggest!!

Use DMSO and read immediately.

in my lab we always use DMSO. we use 200ul of DMSO per well of 96 well plate but I think 100ul will work just as well. After I have added the DMSO to all the wells (you will see the formazan start to dissolve right away), I immediately set the multi-channel pipettor I just used to add the DMSO to half of the volume of DMSO and mix all wells, from lightest to darkest (I set my plate up so the lightest wells, PAO all dead control are at one end, and darkest untreated control most living at the other end, so I don't have to change tips as I go) mix by pipetting up and down 3-4 times per well. Then read immediately. Should get tight numbers unless something is very off. Also I have noticed that if you do NOT read right away, the readings will start to vary a lot more. It seems if you leave it very long the color intensity starts to vary in random ways, so read it immediately. We also have a trick to remove any bubbles - to blow hot air on the samples with a hair dryer, immediately pops any bubbles. But DMSO does not hold bubbles very long anyway, and I set it up in the windy fume hood, so usually no problem having bubbles. But you do want to make sure there are no bubbles in any samples as that can really alter the reading.

I've not used the SDS or any water-based method in my lab, and for our student who went to another lab for the summer and introduced using DMSO, the PI was greatly impressed by the much better results.