PT  - JOURNAL ARTICLE
AU  - Patrick Renner
AU  - Michael Crone
AU  - Matthew Kornas
AU  - KimAnh T. Pioli
AU  - Peter D. Pioli
TI  - Intracellular flow cytometry staining of antibody-secreting cells using phycoerythrin-conjugated antibodies: pitfalls and solutions
AID  - 10.1101/2022.01.10.475671
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.01.10.475671
4099  - http://biorxiv.org/content/early/2022/01/11/2022.01.10.475671.short
4100  - http://biorxiv.org/content/early/2022/01/11/2022.01.10.475671.full
AB  - 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.Competing Interest StatementThe authors have declared no competing interest.