Preparation of uniform haemoglobin free human erythrocyte ghosts in isotonic solution.
A method is described for the preparation of haemoglobin free human erythrocyte ghosts in isotonic solutions using dielectric breakdown technique. In this single haemolytic procedure, almost complete removal of haemoglobin (less than or equal to 0.1%) was achieved by subjecting the erythrocytes suspended in phosphate buffered, isotonic KCl solution at 0 degrees C to three consecutive electrical field pulses of 16 kV/cm in the presence of 10 mM EDTA; EDTA was used to prevent electrical haemolysis. Haemolysis is induced by subsequent dilution with isotonic and isoionic solution to lower the EDTA concentration. Haemolysis is complete after 5 min; the cells are centrifuged, washed and resuspended in a solution of the same composition and osmolarity containing 4 mM MgCl2, but no EDTA. The resealing process, carried out at 37 degrees C, was complete in about 1 h. Measurements of the size distribution of the ghost cells in the hydrodynamically focusing Coulter Counter at varying field strengths in the orifice revealed that the ghost population is nearly uniform. The mean (modal) volume of the ghost cells was 110--120 micronm3 when suspended in phosphate buffered NaCl solution. The apparent breakdown voltage was about 1.3 V.
D004910:Erythrocyte Membrane / Q000032:analysis*; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D007552:Isotonic Solutions; D008722:Methods
D007552:Isotonic Solutions