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MTT assay - need opinions on the procedure (Apr/22/2008 )

Hi all,

Would like some opinions from the pros in MTT on the issue below.

From what I've read on MTT protocols so far, it seems that the general recommendation is to add MTT to cells, then incubate for 4h before adding a solvent such as DMSO to solubilize the formazan crystals.

For my experiment, I would like to subject my cells (H9C2) to chemical hypoxia using CoCl2 for 24hr.
I'm now considering between the 2 options below.

1. a. Add CoCl2 to my cells and incubate them for 24h.
b. remove the CoCl2-containing medium; wash cells with PBS.
c. replace with fresh serum-free medium.
d. add MTT; incubate for 4h.
e. add DMSO, measure OD


2. a. Add CoCl2 to my cells and incubate them for 20h.
b. directly add MTT; incubate for 4h. (making a total incubation time of 24h)
c. add DMSO, measure OD

Which is generally recommended? Is there any difference between the 2 protocols?
Has anyone done MTT on h9c2 cellline?

Thanks guys!


I'm not familiar with CoCl2, or with your specific cell line, so I can't say whether swapping media before running the MTT assay would be a good or bad idea. I can say that my lab just leaves the media in with the compounds we test and adds the MTT assay to that.

We get our MTT from a kit, and the kit instructions say to optimize the incubation periods, both with your compounds of interest and with how long you leave the MTT in the media before adding detergent. Either way you decide to go, you should see if you need the full four hours, or if one, two, three, or sometime in between those will do.


Neither I have worked with you cell line or with CoCl2.
- we add MTT directly to the wells, without removing the medium. we remove the medium only if the colour of the compound we're testing can interfere with the assay.
- we don't include the incubation time of MTT in the 24h treatment
- for some cell line (adherent) we incubate MTT for 2h

so I suggest
- try your second protocol but add MTT after 24h
- you should also see, as TheSquire says, if you can shorten the lenght of incubation of MTT

we prepare MTT by desolving the powder (Sigma) 0.5% in PBS.


I have neither worked with H9C2 cells nor CoCl2.

The protocol of MTT assay I use is as follows:

1. After any treatment, aspirate medium (in your case, CoCl2 containing medium.
2. Wash once with 1xPBS.
3. Incubate cells with 1mg/ml MTT in PBS for 3-4 hours at 37°C. Time can be decreased if you increased the concentration of MTT.
4. Add DMSO (or ispropanol) and measure.

-Franz K.-

You maybe want to have another assay to make sure MTT does not over- or underestimate your viability. MTT needs to be reduced by cells. Cells under hypoxia condition may be over- or hyper reactive for MTT.


[quote name='shyn' date='Apr 22 2008, 08:34 AM' post='133469']
Hi all,

Is there anyboby who is doint MTT assay with M NFS 60 cell lines

Need help and suggestions

Thanks and Regards

-nazia sulthana-