I am evaluating a cytotoxicity effect of a drug based on MTT assay.
I am using the MTT kit of ROCHE for 96 well plate .
Now I'd like to do MTT assay using 6well plate instead of 96 well.
I intended to modify a new protocol from the original protocol as following:
1. A549 cells seed at 5 x10^5 cell/2ml/well (6 well plate)
2. culture for 2 days until 70-80 % of confluence
3. Treated by a drug for 2 days
4. Add 200 μl of MTT reagent per well and then incubate for 4hrs
5. Add 2 ml of solubilization solution in each well and then incubating for overnight
6. pipetting the adherent cells and picking-up them into 96 well plate for reading absorbance.
Finally, total volume will be 4.2ml/ well. Is is too much?
Is there any missing in my protocol? and I am wondering that whether I should remove the culture medium before adding the MTT reagent.
Is there anyone have any procedures that guide me?
Thanks in advance!
Tran Dat
Well the procedure steps seems OK, but I'm not sure about the volumes and concentrations of reagents that you are adding to the wells. That simply needs to be performed in some old plates - to see the volume.
Also I'm not sure about the overnight incubation with destining solution....I dont know what you are using but in my case it usually takes 10 min to dissolve all crystals.
For the overnight incubation you do not need to wait 2 days before the drug treatment. 24 h is enough! and you do not need a long incubation for crystal dissolving, I think that this could affect metabolic activity of cell tested (and interfere with your treatment).
Dear Tran Dat did you make any optimizations of this protocol, and was it successful? Till now I was preforming the MTT test (ROCHE kit) on 96 well plate, but I changed the conditions and I started using 6 well plate. I was not planning to pippet the cells from 6 to 96 well plate..but I consider it as s good idea. I was planning to measure the absorbance in the 6 well plate, rather then 96..
Regarding the time for incubation with the solubilization solution in the MTT kit- I'm doing the ON incubation, too... for my cells 10 min is not enough at all..
Thank you in advance!
Dear Elena Krachmarova,
I got the success of MTT assay on 6 well-plate with a modified protocol using Cell Proliferation Kit (MTT) ROCHE.
In my case, I cultured cells for 2 days with an inhibitor and then aspirated supernatant. Added 1 ml of a fresh medium culture including the MTT labeling reagent (0.5 mg/ml) to each well and incubated for 3 additional hours. Subsequently, 1ml of Solubilization solution was added into each well and allowed the plate to keep during an overnight at 37 oC. The solubilized formazan products were diluted 3 times with PBS or Solubilization solution (It'd better to measure an original concentration before dilution, and then I will decide how many time of dilution should be suitable) and then transferred to 96 well-plate to measure absorbance at 550 nm with a reference of 650 nm.
good luck!
P/s: If you have any further questions, please email me at [email protected]
Dat
Dear Tran Dat , thank you very much! I'm planing to do the same, but since I have ELISA reader that can read 6 well plates I think to stick to the same plate, and not transfer the solubilized formazan product to a 96 well plate.
Your answer was really helpful!
Good luck to you, too!
Best,
Elena
Hi
For 6 well plate what is the amount of MTT assay to be added per well?
Viral Maru , I added 200 microl MTT in the 6 well plate, and after 4 hours I added 2 ml of solubilisation solution.
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