I am trying to figure out the best protocol for lysing neutrophils after a gradient isolation procedure from whole blood. If anyone could pass along a protocol that has worked well I would greatly appreciate it. Thanks!
I didn't understand your question. Are your cells contaminated with neutrophils after gradient and you want to get rid of them? Or have you isolated neutrophils and you want to lyse them?
Hi Madeline,
most often it is necessary to lyse contaminating red blood cells upon density gradient purification of neutrophils, whether you have used Ficoll, polymorphprep or any other method. We commonly use hypotonic solution as detailed below.
1. Purify your neutrophils, wash in PBS.
2. To the cell pellet (containing RBCs and neutrophils) add 5 mL of 0.2% NaCl. Resuspend and incubate 1 min RT.
3. Add 5 mL 1.6% NaCl (will bring it back to physiological salt concentration), mix gently, and spin down the cells according to your usual protocol.
4. Discard the lysed cells, resuspend neutrophils in PBS/medium and proceed by counting/analyzing your purified neutrophils.
This is a gentle protocol which should allow you to get ride of most contaminating RBCs while keeping your neutrophils intact and non-activated.
Please let me know if you have any other questions regarding neutrophil isolation or functional assays.
Best regards,
Christian
Thank you Christian - I sent you a message following up.
Andre - to clarify, I would like to lyse isolated neutrophils for western blotting.
Please appropriate answer for the question above "" I would like to lyse isolated neutrophils for western blotting. ""
This paper has a section in the methods on how to lyse neutrophils for WB. I used to do this very successfully in the lab, it gives you clean protein bands. Make sure your protease inhibitors are fresh or you'll get smeary bands
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Danielle Dunlea I am grateful for your help. I will go through it and try it with mouse bone-marrow derived neutrophil. Thanks
Danielle Dunlea It seems cell fractionalization was is done bcos i noticed different part of the neutrophil (like cell memebrane, cytosol) was used for WB. the entire cell was not lysed together in the paper. Is that the way you adviced too?
Also, Have you used that approach for Mouse bone marrow derived neutrophil bcos I noticed from many lysis buffer i have tried that human and mouse neutrophil behave differently. Thanks for your anticipated response.
Hi Babamale, I only worked with human neutrophils so I cannot answer for mouse bone marrow. I used the same buffer for neutrophil whole cell lyses and ran on western blot without any issues.