RBCs were removed either magnetically using the EasySep™ RBC Depletion Reagent or using ammonium chloride lysis (control). To perform magnetic RBC depletion, whole blood was diluted 1:1 with buffer (phosphate buffered saline plus 2 mM EDTA), the EasySep™ RBC Depletion Reagent added and the sample placed in an EasySep™ magnet for 5 min. Magnetically labeled RBCs were retained in the magnet and TNC were simply poured or pipetted off. The Reagent addition, magnet incubation and pour-off steps were repeated once more, followed in some cases with one more final magnet incubation and pour-off. The number of TNC recovered were counted and the samples evaluated by flow cytometry for RBC content (CD45- / Glycophorin A+) and viability (7AAD-).
4.2±1.3×106 TNC per mL of starting whole blood were recovered when using the EasySep™ RBC Depletion Reagent, compared to 5.5±2.1×106 TNC recovered from the same samples by ammonium chloride lysis (n = 8; p <0.05). RBC contamination of the TNC fraction was significantly lower when samples were treated with the EasySep™ RBC Depletion Reagent rather than ammonium chloride (1.2 ± 1.3% versus 19 ± 18% of total events were CD45- / Glycophorin A+ respectively; p <0.05). The viability of the TNC obtained by either method was 99 ± 1%.
RBCs can be immunomagnetically depleted from a sample of whole blood in 10 - 15 minutes, without exposing the cells to hypotonic lysis or the need for hetastarch sedimentation. The isolated nucleated cells are immediately ready for analysis or further downstream assays.
C.E. Peters:Employee; Company/Organization; STEMCELL Technologies Inc.T.N. Lee:Employee; Company/Organization; STEMCELL Technologies Inc.S.M. Woodside:Employee; Company/Organization; STEMCELL Technologies Inc.T.E. Thomas:Employee; Company/Organization; STEMCELL Technologies Inc.A.C. Eaves:Other (Identify); Company/Organization; Owner STEMCELL Technologies Inc.