You don't mention your source of buffy coat : normal donor peripheral blood, cytokine stimulated normal donor or patients peripheral blood or monoculear products obtained from apheresis. It has been my experience that erythrocytes that are 'that' resistent to lysis are usually immature nucleated RBC and will not lysis! (also will be enumerated in electronic cell counters as small lymphocytes) Have you looked at these cells under a microsope to determine if they are mature or not? I have had good luck using ~1.2%(w/v) ammonium oxalate solution (210mOsm) for lysing mature RBC. We chose this after extensive comparisions of hypotonic, ammonium chloride, etc. The ammonium oxalate was the least damaging when cells were evaluated morphologically, ie minimal vacuolation, membrance damage. These cells were also evaluated functionally : lymphs (mitogenesis), monocytes (phagocytosis), granluocytes (chemotaxis & phagocytosis), and platelets (aggregation) and all responded within normal ranges, while ammonium chloride activated lymphocytes. If you would like to try our lysing solution I will be happy to share our SOP with you. April Translational Research & Flow Ctytomerty Blood & Marrow Transplantation Univ Texas MD Anderson Cancer Center
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