We are stimulating human neutrophils with priming agents and chemoattractants such as C5a, with the neutrophils suspended in 1% serum and RPMI with HEPES. This is being conducted in a standard eppendorf tube. We find on subsequent flow cytometry that the numbers of neutrophils falls markedly relative to control treated cells, and suspect they may be sticking to the sides of the tube. Does anyone have any experience of this, and how did you deal with it? Any role fo low-adherence eppendorf tubes?
Hello Andrew,
How long is the stimulation protocol? If the concentration of priming agents, or the incubation time, is too long, you may be seeing large amounts of cell death (showing up as debris in FC, rather than dead/apoptotic by viability staining). If you are concerned about adherence, you can (1). Increase the serum concentration to 5% and/or (2) do a brief wash with EDTA before removing cells from the tubes or plate. This should also halt their activation.
Best,
Meaghan R.
Hi Andrew,
I agree with Meghan. When it comes to neutrophils, the culture duration is really important . I have experience working with murine neutrophils/monocytes/bone marrow derived macrophages. Neutrophils (esp. after stimulation) start dying at around 6 hours and very few are alive at 24 hours after culture. The loss you are seeing could be from the cells dying, rather than sticking to the culture tubes. You could always use low-adherence eppendorf tubes, just to be sure.
Hi both, many thanks for your answers - we are stimulating the neutrophils for 1 hour with C5a - certainly I would agree that at prolonged incubations we see more cell death (and at 6 hours see a distinct drop off in cellular activity which is probably a pre-cursor to death despite not being positive on live/dead stain).
We will try EDTA and Trypsin to see if we can lift cells from the sides of the vessel .
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Medical Pharmacology
- Clinical Pharmacology