In our lab, we generally use 3 rounds of 10 minute, 4* incubation with tris ammonium chloride (Stem Cell Technologies) for red blood cell lysis of murine blood.
However, neutrophil counts by flow cytometry after this preparation generally yield lower results than one would expect, and I'm concerned we may be preferentially killing neutrophils during this stringent lysis.
Have neutrophils been shown to be more sensitive to RBC lysis buffers? If so, do you have any recommendations for a less stringent lysis procedure that will still yield adequate RBC clearing?
We have previously used dextran sedimentation followed by Ficoll separation and a final step of RBC lysis using water for approx 25 seconds. The suspension was then equilibrated using 10xPBS followed by a final centrifugation step. Whist this provided us with 'happy' neutrophils, the purity was approx 95%. We have since moved to neutrophil isolation kits which negatively select for neutrophils from whole blood. These are much cleaner preparations.
We have previously used dextran sedimentation followed by Ficoll separation and a final step of RBC lysis using water for approx 25 seconds. The suspension was then equilibrated using 10xPBS followed by a final centrifugation step. Whist this provided us with 'happy' neutrophils, the purity was approx 95%. We have since moved to neutrophil isolation kits which negatively select for neutrophils from whole blood. These are much cleaner preparations.
I am not familiar with murine blood. Is there a reason not to do a one step lysis with any available ammonium chloride and just use markers for gating on neutrophils?
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