Anti cd45ra bv421

Cells were washed in PBS + 10% FCS and stained with primary antibodies (anti-CD45RA-BV421, anti-CD62L-APC-Cy7, anti CDCD49f-FITC, anti-CD51-APC, anti CD493-FITC, anti-CD49b-APC, anti-CD49a-APC, all Biolegend; anti-IL17a-Alexa647 and anti-CD146/MCAM-PE, both BD Bioscience) for 30 min on ice. Intracellular staining of IL-17a was performed by implementation of the CytoFAST Fix/Perm buffer set (Biolegend) exactly according to the manufacturer’s instructions. The cells were fixed in 4% PFA (Sigma) and the stainings were assessed on a BD Canto II flow cytometer.

Flow cytometry studies of the single cell suspensions were carried out with a BD LSRII-Fortessa™ system as previously described (Feng et al., 2011 (link)). The cells (100 µl of cell suspension including 10⁶ cells) were blocked for 5 min at room temperature (with mouse serum for rat cells, or rat-anti-mouse CD16/32 for mouse cells). Cells were then incubated on ice for 30 min with an antibody panel. For rat cells, the following antibody panels were used: CD3 (clone 1F4 BV421-labeled, 1/500, for total T cells), CD4 (clone OX-38, FITC-labeled, 1/500, for CD4 T cells), CD8a (clone OX-8; PE-labeled, 1/500, for CD8 T cells), and CD45RA (clone OX-33; BV786-labeled, 1/500, for B cells) (all from BD Pharmingen, San Diego, CA). Ghost Dye™ Red 780 was used to detect the live and dead cells (Tonbon Bioscience, San Diego, CA). Cells were then washed once with fresh buffer (PBS/2 mM EDTA/2% FBS) and ran flow cytometry freshly. For mouse samples, anti-CD3-APC (for total T cells), anti-CD4-FITC (for CD4⁺ T cells), anti-CD8a-PE (for CD8⁺ T cells), anti-CD45RA-BV421 (for B cells) (all from BioLegend) were used. Flow cytometry data were analyzed with FlowJo software (Ashland, OR). Cell numbers were calculated from the frequency of each positive staining against the total cell number in the sample.