Fluorescently labeled dengue viruses as probes to identify antigen-specific memory B cells by multiparametric flow cytometry

Low frequencies of memory B cells in the peripheral blood make it challenging to measure the functional and phenotypic characteristics of this antigen experienced subset of B cells without in vitro culture. To date, reagents are lacking to measure ex vivo frequencies of dengue virus (DENV)-specific memory B cells. We wanted to explore the possibility of using fluorescently labeled DENV as probes to detect antigen-specific memory B cells in the peripheral blood of DENV immune individuals. Alexa Fluor dye-labeled DENV yielded viable virus that could be stored at -80 degrees C for long periods of time. Using a careful gating strategy and methods to decrease nonspecific binding, we were able to identify a small frequency of B cells from dengue immune individuals that bound labeled DENV. Sorted DENV+ B cells from immune, but not naive donors secreted antibodies that bound DENV after in vitro stimulation. Overall, Alexa Fluor dye-labeled DEN Vs are useful reagents to enable the detection and characterization of memory B cells in DENV immune individuals. (C) 2014 Elsevier B.V. All rights reserved.