We are currently using a commercial ammonium chloride based lysing agent (PharM Lyse, BD Pharmingen) for peripheral blood, PBPC, bone marrow and cord blood. 100uL of sample containing 1e5 - 1e6 nucleated cells is stained with directly conjugated mAb for 15min at ambient temperature (protect from light) For PB, BM, CB : add 2mL of 1X PharM-Lyse, incubate 5min, ambient (protect from light) Add 2mL of PBS+1%serum; sediment cells 200g for 5min, ambient temperature. Repeat procedure if all RBCs are not lysed. Note : nucleated RBCs will not lyse For PBPC : add 500uL of 1X PharM-Lyse, incubate 1.5min, ambient (protect from light). Add 3mL of PBS+1%serum; sediment cells 200g for 5min, ambient temperature. april April G. Durett, MSc Research Associate Clinical Applications Laboratory - Flow Cytometry Center for Cell & Gene Therapy Baylor College of Medicine Phone 832.824.4666 (voice mail) FAX 832.825.4668 e-mail agdurett@txccc.org -----Original Message----- From: Rudensky [mailto:rudensky@szmc.org.il] Sent: Tuesday, February 20, 2001 4:43 AM To: cyto-inbox Subject: BONE MARROW LYSIS we are presently processing bone marrow for facs analysis by ficoll , and peripheral blood by direct lysis . i would greatly appreciate receiving someone's successful protocol for lysis of bone marrow
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