5 replies to this topic

[shs]

Hi all  

I treated the Huh-7 (hepatocarcinoma) with MTT several times but what I got was crazy, there is no difference between the untreated cells and the cells treated with different doses of drugs (until now I tested 3 dhfferent drugs but I faced the same problem) the protocol I use is the following:
1- Plate the cells out in 96 wells and incubate them for 24 hours (cell density 15X103/well(200ul))
2- Aspirate the media and treat the cells with 200ul of drug that dissolved in serum free media) and incubate for another 24h
3- Add MTT (dissolve 0.025mg/5ml PBS) and incubate for 4 hours
4- Aspirate the media and dissolve the formazan by adding 200ul DMSO

The problem is when I check the cells under the microscope I could see the difference in formazan precipitate but after adding DMSO the purple color doesn't reflect what's expected (sometimes the absorbane of untreated cells is lower than the cells treated with the highest conc. of drug   ).
I optimized the cell density and the incubation time as well.

Can anyone tell me how to do positive control (in details plzzzz)
I'm absolutely new in this field and ur help will be appriciated.

[shs]

OMG No help

[dw237606]

Have you tried using media that contains serum?  In the past I have had problems with certain cell lines when using serum free medium but I have no experience with this cell line.

Are you using 0.025mg/mL of MTT or 0.025 mg in 5 mL (making it 0.005 mg/mL)?  I highly doubt this is the source of the problem but I have always used 3-5 mg/mL of MTT, and put 50 uL of the MTT solution along with 100 uL of complete media and allowed it incubate for 4 hours before aspirating and stabilizing in DMSO.  

For a positive control the standard way that I have seen done is as follows - this is probably the way you are already doing it but just in case I'll outline the procedure here in detail:

Plate the same number of cells in the wells as you are for the wells that will receive treatments.
After 24 hours aspirate the medium the same as all the other wells, and replace it with with fresh, complete medium.
After incubating for however long, aspirate the medium (same as all the other wells) and replace with the medium/MTT solution describe above.
Incubate for 4 hours, aspirate the medium/MTT solution, solubilize the dye in DMSO (100 uL) and then add 15 uL of 0.1 uM glycine buffer (pH 10.5)
Read the absorbance.

[dw237606]

I'm sure you have already thought of this and I don't mean to insult your intelligence but something else that might be worth trying is solubilizing the formazan in 100 uL of DMSO instead of 200 in order to magnify any changes in dye content in each well.  

What are the 3 drugs you have tried and at what concentrations?

[bknm]

I always dissolve in 100ul DMSO. Also do you have a positive control that is known to kill the cells? I usually use a small bit of diluted tween to ensure I get killing of cells and that my MTT/DMSO reaction is working.

[shs]

Thank you all