In an LDH cytotoxicity assay, what to interpret if compared to control cells, my cells show significantly less LDH in supernatant after normalization with protein. I am working on MCF-7 cells as control and VEGF siRNA transfected cells as treated.
Hi Heta,
check your assay setup and caluculations. Also make sure that you treat all cells absolutely the same way (Medium change, cell number, mock transfection.....).
Some questions:
Have you transfected your control cells with empty vector? If not, the presence of transfection reagent might interfere with the assay.
How long did you treat your cells? If the period of treatment is long, a difference in growth rate could lead to these results.
What kind of "normalisation with protein" did you perform? To my knowledge, usually equal cell numbers are seeded and the LDH release of this defined cell number is analysed afterwards!
Are you using a comercial kit or a "self-made" LDH assay? Check troubleshooting sections of commercial kits, as these often provide helpful advices.
More details might help to sort out your problems!
Best,
Kai
Kai F. Albring THANK YOU.
that was very helpful. Regarding assay conditions, incubation period is 24 hours. I use accurex biomedical kinetic assay kit for estimating supernatant. Yes, transfection with empty vector is done which shows LDH result intermediate between control and VEGF-c siRNA transfected. Simple protein estimation from cell lysate was performed by bradford method. even if we dont consider protein estimation, results for LDH are same.
Yuan Gu - did you find any logical explaination for this phenomena?
Elodie Louvier - I have added 10000 cells / well after counting only.
In a normal culture condition (control group), LDH can also be released by cells into medium. This can be evidenced by comparing the OD value of 'control' group with that of 'only medium' group. Under untoxic condition (no cell death), the OD value is theoretically proportional to the number of cells.
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