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

BACKGROUND: 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 ASCs regulate multiple aspects of biology through the secretion of various cytokines. In this regard, ICFC is a key tool used to assess the presence of intracellular proteins such as cytokines and transcription factors. METHODS: Paraformaldehyde plus saponin or the eBioscience Foxp3/Transcription Factor Staining Buffer Set were used to evaluate the non-specific intracellular retention of phycoerythrin-containing antibody conjugates by ASCs. RESULTS: We showed that the use of phycoerythrin-containing antibody conjugates led to a false interpretation of ASC intracellular protein expression compared with other cell types. This was mainly due to the inappropriate retention of these antibodies specifically within ASCs. Furthermore, we demonstrated how to reduce this retention which allowed for a more accurate comparison of intracellular protein expression between ASCs and other cell types such as B lymphocytes. Using this methodology, our data revealed that spleen ASCs expressed toll-like receptor 7 as well as the pro-form of the inflammatory cytokine interleukin-1β. CONCLUSION: Increasing the number of centrifugation steps performed on ASCs post-fixation leads to inappropriate retention of phycoerythrin-containing antibody conjugates during ICFC.