Anti cd45r b220 fitc clone ra3 6b2

Spleens were harvested in Flow Cytometry Staining Buffer (eBioscience) and cell suspensions were made by expressing spleen tissue through a cell strainer using the plunger of a 10 ml syringe. Cell suspensions were then washed, centrifuged and resuspended in 1 ml of the buffer. Splenocytes were counted on a hemocytometer and 5 × 10⁵ cells were incubated with Fc-block (anti-mouse-CD16/CD32 antibody; eBioscience). Samples were stained with the following antibodies: anti-CD45R (B220)-FITC (clone RA3-6B2; BD Biosciences), anti-CD4-FITC (clone GK1.5; BD Biosciences), anti-CD8-PE (clone 53-6.7; BD Biosciences), and isotype-specific control anti-rat IgG2a κ-FITC and -PE antibodies (clone R35-95; BD Biosciences). All antibodies were diluted to 0.5 μg for 5 × 10⁵ cells. Splenocytes (10,000 events/sample) were analyzed using a FACSCanto flow cytometer with FACSDiva software (BD Biosciences). Data analyses were conducted with FlowJo software (FlowJo). Dot plots of forward scatter (FSC) vs side scatter (SSC) were used to exclude debris, and then doublets were excluded using FSC-height (H) vs FSC-width (W) and SSC-H vs SSC-W plots (Supplementary Fig. 8). The number of each subset was calculated as the percentage of labeled cells multiplied by the total number of splenocytes.