Intracellular flow cytometry staining of antibody-secreting cells using phycoerythrin-conjugated antibodies: pitfalls and solutions

Abstract Antibody-secreting cells are terminally differentiated B cells that play a critical role in humoral immunity through immunoglobulin secretion along with possessing the potential to be long-lived. It is now appreciated that antibody-secreting cells regulate multiple aspects of biology through the secretion of various cytokines. In this regard, intracellular flow cytometry is a key tool used to assess the presence of intracellular proteins such as cytokines and transcription factors. Here, we showed that the use of phycoerythrin-containing antibody conjugates led to a false interpretation of antibody-secreting cell intracellular protein expression compared to other cell types. This was mainly due to the inappropriate retention of these antibodies specifically within antibody-secreting cells. Furthermore, we demonstrated how to reduce this retention which allowed for a more accurate comparison of intracellular protein expression between antibody-secreting cells and other cell types such as B lymphocytes. Using this methodology, our data revealed that spleen antibody-secreting cells expressed Toll-like receptor 7 as well as the pro-form of the inflammatory cytokine interleukin-1β.