My response to a query regarding RBC lysis drew several requests for our procedure, so thought I would share with everyone. I came upon this technique when setting up platelet antibody assays; used it latter to isolate various cell populations (platelets, lymphs, mono, grans) from whole blood and apheresis collection products during development of blood cell separator with IBM. Before choosing this lysing agent we tested several methods and found this reagent to be the least traumatic to all cell populations, by both morphological evaluation and functional assays. Original Reference : Aster, RH, HE Cooper, and D Singer, Simplified Complement Fixation Test for Detection of Platelet Antibodies in Human Serum; J Lab Clin Med 63: 161-172, 1964 (January) Current Procedure for Preparation and Use Reagents 1. Ammonium oxalate, oxalic acid Source : Sigma Chem, St Louis MO, USA, #O-8751 2. Sterile water for irrigation, USP Source : Baxter Heathcare, Deerfield IL, USA, #2B7114 This is more for our convenience, any triple distilled water source is acceptable. Preparation 1. Prepare 1.1-1.2%(w/v) ammonium oxalate in water 2. Mix well until reagent is in solution 3. Adjust solution to 205-215mOsm; this is critical to achieve RBC lysis. 4. Filter sterilize using 0.2um Corning Filter/Storage unit 5. Store aliquots at room temperature or store 4C, bring to ambient temp prior to use RBC Lysing Procedure 1. Dilute 1 part sample : 4-5 parts ammonium oxalate Note : sample = peripheral blood, bone marrow, cord blood, apheresis products, lymph/mono interface or RBC/gran pellet from Ficoll gradient separation. 2. Mix well by gentle inversion; incubate 15 min ambient temperature 3. Sediment cells, 400g 8min, 25C 4. Decant wash supernatant containing free hemoglobin 5. Gently resuspend cell pellet 6. Repeat steps #1-#6 if necessary; maybe repeated up to 3X without damaging cells. Red cells remaning after 3 lysing procedures are more than likely nucleated RBC and will NOT lysis 7. Wash cells in PBS+1%(v/v)HSA, or diluent of choice according to protocol for cell useage. If there are any further questions, please contact me April Translational Research & Flow Cytometry Blood & Marrow Transplantation Univ Texas MD Anderson Cancer Center at Houston
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