I want to check the cytotoxicity of my material. I am using MTT assay kit. The protocol is provided in the booklet with kit how to perform the assay. Is it better to follow the protocol provided in the kit or use protocol given in literature review. As in literature review different protocols are given performed with different kits.
Hi Noor,
no kit necessary:
· seed cells in 150µl medium into a 96well plate, add your inhibitor(s)
· add 10µl 0.5% MTT in PBS (e.g.Sigma M2128) = 5mg/ml for a final concentration approx. 0.3mg/ml
· incubate for 4 to 5h to o/n
· add 100µl 10%SDS in 0.005M HCl (= 1:2000 conc. HCl)
· incubate o/n @ 37°C and read 550nm (reference wavelength more than 650nm)
If you have the kit already - follow their instructions!
Best wishes
Robert
Hello Noor Ul Ain
Since you are using the MTT assay kit, please follow the protocol provided in the kit.
Usually, in the MTT assay kit the MTT reagent powder is provided. You will have to prepare the MTT reagent as per the kit’s instruction namely, adding the required volume of cell-based assay buffer in the MTT vial and completely dissolving the powder. The MTT powder may dissolve slowly in the buffer. Vigorous vortexing will be needed to dissolve the powder completely. MTT solution appears bright yellow in color. You may add 10ul (or the required volume as mentioned in the kit’s protocol) of the reconstituted MTT reagent per well.
If you do not consume the MTT reagent in a single experiment, you need to store the reconstituted vial at -20 degree C in amber colored bottle until further use.
Different MTT kits have different protocol, but the objective remains the same (i.e., measuring cytotoxicity). You may have different protocols in the literature for MTT assay which may be designed as per one’s needs. For example, in the MTT assay you may use any one of the solubilization solution (like DMSO or acidified isopropanol solution, or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid) to dissolve the insoluble purple formazan product into a colored solution. The solubilization solution is also provided in the kit (the composition of which is not disclosed due to proprietary information) which you will have to use.
Since you have the MTT assay kit available with you, it is best in such a situation to follow the protocol provided in the kit.
Best.
Robert K Slany Thank you for your advice. I have MTT assay kit in the lab so I want to make sure whether to use kit protocol or follow the literature and there are different methods provided.
Malcolm Nobre I am using Ab cam MTT assay kit and MTT reagent is in solution form already. It says that I should add 50 ul of MTT reagent and 50 ul of media. But I don't know the concentration of MTT reagent in mg/mL. It is not given. How can i determine that?
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