I am doing LDH assay for the THP-1 cells infected with the Salmonella to assess pyroptotic cell death. I am using different bacteria concentration in RPMI medium supplemented with FCS and lyse uninfected cells with 1% tryton X100 for the positive control (high death). I can see that the lysed cells are totally dead, while infected cells are partially looking alive. But I got higher color reaction in infected cells than in lysed cells. The only difference was in the cell medium (I used up the aliquote for the bacteria dilution, so I had to take another older one for the positive and negative controls).
Does anyone have any experience with medium age / pH / storage conditions affecting LDH assay activity?
Hi,
what about LDH activity introduced and excreted by the bacteria? If you compare uninfected and infected cells and cannot remove the bacteria from the wells completely, you will have bacterial LDH contributing to the total signal in addition to the cells' LDH.
Another source of additional activity may be the serum supplementation, however, you can correct for this with appropriate controls and blank measurement.
We have encountered something similar in wells unintentially contaminated with bacteria in the MTT assay, which surprisingly are also able to reduce MTT very efficiently (signals two to three times over controls).
Hi,
Use freshly collected media or lysate samples to assay the amount of LDH released into the media or contained within the cells.It is possible that the Triton X gives a background color, which has to be deduced in order to accurately reflect the value of lysate. If Triton X alone (negative control) gives a color reaction with the reagent, you may not consider lysing the cells in Triton X.
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