BD Biosciences
PE Rat Anti-Mouse CXCR5
561988
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The goal of this experiment was to identify T follicular helper cells (Tfh) by flow cytometry using canonical markers such as CXCR5 and PD-1. Unfortunately, the CXCR5-PE antibody did not provide good staining to distinguish positive cells from background. Will have to try two-step staining with CXCR5-bioitin + Streptavidin.
Flow Cytometry
Splenocytes
30 mins at +4C.
Anti-CD16/CD32
NA
LSR II
CXCR5 is expressed on B cells but I did not observe a good staining on CD3 neg splenocytes. With further gating on CD3+CD4+ T cells, Tfh cells were supposed to be PD1+CXCR5+ but I did not observe a positive population. My observations are in line with previous report by this article http://www.nature.com/protocolexchange/protocols/2707#/troubleshooting The authors recommended a two-step staining strategy with CXCR5-biotin + Streptaividin-PE/BV421/APC.
None
Poor separation of positive and negative cells
Try CXCR5-biotin.