Direct immunomagnetic depletion of RBC without hypotonic lysis or sedimentation.

Abstract

Abstract Isolation of specific cell types for functional studies frequently requires red blood cell (RBC) depletion as a first step. However, hypotonic lysis of samples may be deleterious to certain cell types, and RBC removal by hetastarch sedimentation is inconsistent. Furthermore, RBC depletion with agents such as ammonium chloride (NH4Cl) is often ineffective, leaving samples that still contain many RBC. We have developed an immunomagnetic method to deplete RBC that is rapid, can be used with a variety of sample types, and leaves untouched total nucleated cells (TNC) ready for downstream assays. RBCs were removed from four different types of samples (whole blood, leukopak, buffy coat and bone marrow) either magnetically using the EasySep™ RBC Depletion Reagent or by NH4Cl lysis. To perform magnetic RBC depletion, samples were diluted 1:1, the reagent added, and the sample placed in an EasySep magnet. Magnetically labeled RBC were held in the magnet and TNC were poured or pipetted off. The reagent addition, magnet incubation and removal steps were repeated a second time, and in some cases the magnet incubation and removal steps were repeated a third time with whole blood and buffy coat samples. RBC contamination of the TNC fraction was <2% for whole blood (n=8), leukopak (n=2), and bone marrow (n=5) samples treated with EasySep™, and 12 ± 10% for buffy coat (n=4) samples treated with EasySep™; in all cases this was equivalent to or significantly lower than the RBC contamination in the same samples treated with NH4Cl. TNC recovery from samples RBC-depleted by either EasySep™ or NH4Cl lysis was similar. RBCs can be immunomagnetically depleted from samples in 6–15 minutes, depending on the sample type, and TNC are immediately ready for downstream assays.