Using this kit, I have found a greatly reduced luminescent signal in my treatment groups that have bacteria, and I am wondering if the bacteria have the ability to alter the kit reagents.
I don't have experience using this kit but are you sure that the bacteria are not killing your cells? Higher MOIs of bacteria induce higher levels of cell death (depending on the strain of bacteria you use). Maybe try an alternate cell death assay to verify that the reduced luminescent signal you see is not due to bacteria induced cell death. Just keep in mind that some assays are not compatible when you have live bacteria in the culture. We used to do Neutral red assay (and avoid MTT) to measure cell death in response to salmonella infection. You could also measure G6PD release into the supernatants as a surrogate for cell death.
Hello Julie,
Thank-you for your response. For my bacterial infection, I do not see gross cell death by viewing the cells with a cell culture microscope - there are similar numbers of cells as compared to my control wells. I also do not see induction of necrosis with the bacterial infection. For this assay, I do not see an increase in luminescence, indicative of apoptosis, prior to the drop in luminescence, either. Thank-you for the alternative assay suggestions for measuring cell death during infection.
Hi Nicole,
Thanks for the clarification. The other possibilities if you don't see observable cell death under a microscope are (in my opinion)
1. Your bacteria might be interfering with your kit performance.
2. You have better cell survival in your infected wells (hard to see a difference under the microscope when all your wells are confluent). When we used to stimulate cells with low MOI of bacteria (which does not induce cell death), there was an increased Neutral red signal (indicative of increased proliferation/better survival) compared to our unstimulated/uninfected cells (where cells die off due to lack of stimulation). This could explain why you have higher luminescence signal in your control wells. But obviously, this would depend on the strain of bacteria/cells you have.
Alternate cell death assays should help you. One thing I forgot to mention which is super easy is to stain your cells with PI/Hoechst and do fluorescent microscopy.
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