Fast and easy isolation of CD27-positive human memory B cells using EasySep&[trade] Releasable RapidSpheres&[trade

Abstract

Abstract Over the course of a lifetime, memory B cells are key to maintaining antibody-mediated protection from viral and bacterial pathogens and they are one outcome of an effective vaccine. Since memory B cells are typically found at frequencies of less than 5% of normal human peripheral blood mononuclear cells (PBMC), research into their function would be aided by a fast and easy isolation method. To meet this need, we have developed an improved EasySep™ kit for the isolation of CD27-positive (CD27+) human memory B cells from fresh PBMC. First, CD27+ cells are labeled using an antibody cocktail and EasySep™ Releasable RapidSpheres™ and are positively selected using a hand-held EasySep™ magnet. Next, magnetic particles are released from the positively-selected cells, and then non-B cells within the CD27+ fraction are targeted for depletion using a second antibody cocktail and EasySep™ Dextran RapidSpheres™. Following an additional magnetic separation step, the particle-free, CD27+ memory B cells are poured off into a new tube and ready for use. With one additional step, CD27− naive B cells can be isolated from the same starting sample. The entire isolation protocol is completed in under 40 minutes without any centrifugation steps. Using this method, CD19+CD27+ memory B cells can be isolated to 97±2% purity and 37±14% recovery (average of n=19 ± S.D.). The CD19+CD27− naive B cells can be isolated to 93±5% purity and 28±11% recovery (n=9 ± S.D.). When stimulated with CpG and IL-15, the isolated memory and naive B cells are functional, as assessed by both proliferative responses and the secretion of IgG.