Pe cf594 anti cd154

Human PBMCs at 10⁶/tube were stained in duplicate with allophycocyanin (APC)-H7-anti-CD3, BV510-anti-CD4, fluorescein isothiocyanate (FITC)-anti-CD45RA, phycoerythrin (PE)-Cy™7-anti-CCR7, peridinin-chlorophyll proteins (PerCP)-Cy™5.5-anti-CXCR5, PE-anti-ICOS, BV421-anti-PD-1, PE-CF594-anti-CD154, or proper IgG isotype controls (Becton Dickinson, San Diego, CA, USA) at room temperature for 30 minutes. After being washed with phosphate-buffered saline (PBS), the cells were analyzed by flow cytometric analysis using a BD FACSAria™ II (BD Biosciences, San Jose, CA, USA). The data were analyzed with FlowJo software (version 7.6.2, by Flowjo LLC, OR, USA). We analyzed at least 50,000 events per sample and calculated the numbers of different subsets of circulating memory Tfh cells in individual samples according to the counts of lymphocytes per liter of blood multiplied by the percentage of different subsets of memory Tfh cells in lymphocytes.

Human PBMCs at 10⁶/tube were stained in duplicate with APC-H7-anti-CD3, BV510-anti-CD4, FITC-anti-CD45RA, PE-Cy7-anti-CCR7, PerCP-Cy5.5-anti-CXCR5, PE-anti-ICOS, BV421-anti-PD-1, PE-CF594-anti-CD154, or isotype-matched control IgG (Becton Dickinson, San Diego, USA) at room temperature for 30 minutes, respectively. After being washed with PBS, the cells were subjected to flow cytometry analysis in a BD FACSAria Ⅱ (BD Biosciences, San Jose, CA USA) and data were analyzed with FlowJo software (v7.6.2). At least 50,000 events per sample were analyzed. The cells were gated initially on living lymphocytes, on CD3+ and CD4+, and then on CXCR5+ and CD45RA-. Subsequently, the frequency of ICOS+, PD-1+ or CD40L+ CD3+CD4+CXCR5+CD45RA- T (memory Tfh) cells were analyzed. In addition, the CD3+CD4+CXCR5+CD45RA- Tfh cells were gated on CCR7 and the frequency of ICOS+, PD-1+, CD40L+ CCR7+ or CCR7- Tfh cells was analyzed. The numbers of each type of memory Tfh cells in individual subjects were calculated by the counts of lymphocytes per liter of blood multiplied by the percentage of different subsets of memory Tfh cells in lymphocytes.