Brilliant violet 570 cd4

Spleens were removed from euthanized animals, and single-cell suspensions of leukocytes were prepared by disaggregation of the tissue through a 100-μm nylon mesh, followed by dissociation, as we reported previously [39 (link)]. Cells were washed once with complete DMEM media, supplemented with 10% heat-inactivated FBS (hiFBS), then incubated with RBC lysis buffer for 1 min to remove red blood cells, then washed again in complete DMEM media. Cell isolates were prepared, as above, then washed and resuspended in staining medium (1× PBS, 0.5% BSA, 0.02% sodium azide). Ghost Dye Violet 510 was used for live-/dead-cell staining. Leukocytes were labeled with a combination of the following antibodies: PE-Cy5 CD3ε, Brilliant Violet 570 CD4, PE/Cy7 CD8a, APC-Fire750 CD11b, Brilliant Violet 785 CD19, PE/Dazzle 594 DX5, Alexa Fluor 700 F4/80, Brilliant Violet 650 Ly-6C, and Brilliant Violet 711 Ly-6G (BioLegend, San Diego, CA, USA). Gating strategy is as shown in Figure 2a. Data were acquired and analyzed using LSR II flow cytometry and Flowjo software (BD Biosciences, Franklin Lakers, NJ, USA).