I am stimulating mouse bone marrow PMN with PMA in a luminol ROS detection system, and reading the luminescence with a luminometer for 1 hour. I always get two peaks in my curve (an sharp, immediate peak and a broad sustained peak), but I'm not sure why. All the literature I read only has one, sharp peak followed by a steady decline back to zero. Can anyone tell me why this is happening, or suggest ways to get more consistent results?
PMA is quick react and stimulate cells and ideally you should be getting responses almost immediately. The second peak might be independent to PMA and perhaps cells maybe additionally being activated through other pathways such as phagocytosis of dead cells?? macrophages attaching to plates? Perhaps your consistent reading is best measured by deciding on a cut-off point of when to end your assay e.g. 15 min? not 1 hr. You could also check the quality of your cell prep going into the assay.
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