How can I limit this phenomenon in order to have a better negative control?
It is possible that ex vivo neutrophils can release NETs without "apparent" stimuli. Of course the key operative being apparent. I would think improving the speed of initial neutrophil isolation may be a place to start. Working quickly, cold solutions, fresh buffys can all work in your favor.
Thank you for the suggestion! I will try to improve the manipulation steps.
Best,
Rocco
Agree with previous answer, other inadvertant activation causes include exposure to calcium, glass and can occur during red cell lysis. It would be helpful if you could describe your isolation protocol.
Otherwise just make sure you're using buffers without calcium (e.g. HBSS w/o CaCl2). If you're using ficoll/histopaque gradient, don't overload your gradient with blood (this can lead to red cell contamination of your PMN buffy coat, requiring red cell lysis which can activate). Also anecdotally we've observed that anticoagulating blood with K2-EDTA is better than ACD.
Good luck!
Dear Eric,
Hi isolate PMN from fresh blood using Histopaque gradient and then, if there are red cell contamination I eliminate red cells with a lysis buffer. Finally, I use classic RPMI to perform the experiment.
RPMI shoud be supplemented with FCS or the medium alone is sufficient for PMN culture or stimulation?
Best,
R
Hi Rocco,
I also had that problem previously. After receiving lots of suggestion, I could get the very good isolated cells without activation.
I replaced the RBC lysis buffer by distilled water, incubated for only 20sec and than added HBSS 10X to stop the reaction (final concentration of HBSS is 1X). I also reduced the centrifugation speed to 1100rpm at 4oC.
In whole process, I did not pipette up and down (which can activate Neutrophils), just mixed gently.
Moreover, I used RPMI 1640 with 10%FBS and I could maintain the cells for up to 24hr without remarkable cell death and morphology change.
Good luck!
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