Scott Laboratory - Immios Biologics

Mark D. Scott, Ph.D.  • Clinical Professor Emeritus

Pathology and Laboratory Medicine & Centre for Blood Research at the University of British Columbia, Vancouver, BC  Canada

RBC Osmotic Lysis

and Resealing

Osmotic Lysis and Resealing of

Red Blood Cells

Entrapment of exogenous materials (e.g.,  purified α-chains) within normal erythrocytes can be readily accomplished by the osmotic lysis and resealing of normal RBC (human, mouse, etc) as schematically shown. Briefly, washed, packed erythrocytes (80-85% hematocrit) are mixed with the purified CO-α-chains (10 mg/ml packed red cells) and then placed as a thin film within 11.5 mm diameter dialysis tubing (MW cutoff of 3500). The samples are dialyzed against a hypotonic lysis buffer (5 mM potassium phosphate buffer and 2 mM EDTA; pH 7.4) at 4° C for 60 minutes. The dialysis tubing is then transferred to an isotonic resealing buffer (5 mM potassium phosphate, 160 mM NaCl, and 5 mM glucose; pH 7.4) with gentle agitation for 30 minutes at 37° C. Following resealing, cells are washed with saline until the supernatant is clear. Using this procedure, approximately 70-80 percent of the initial packed erythrocyte volume is recovered. Radiolabeled α-chains can be utilized to quantitate the intracellular entrapment.

Scott, M.D. Model Human ß Thalassemic Erythrocytes: Effect of unpaired purified α-hemoglobin chains on normal erythrocytes. In: Beta Thalassemia (Editor: Zakaria, M.), INTECH. Croatia.  ISBN: 978-1-83880-587-6 (2019). In press.  DOI: 10.5772/intechopen.90288

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