Percp conjugated streptavidin

Erythrocyte-depleted spleen or bone marrow cells were stained and analyzed as previously described [31 (link)]. Half a million RBC-depleted splenocytes were incubated with mouse IgG (Sigma-Aldrich) for 15 min prior to staining with various combinations of directly-conjugated mAbs. The following directly conjugated mAbs were purchased from BD Biosciences (San Diego, CA): FITC-conjugated anti-CD86 (GL1), -CD95 (Jo2), -CD4 (RM4-5), -CD3 (145-2C11), -CD44 (IM7), -CD62L (MEL-14), -CXCR5 (2G8), and -GL7 (GL7); biotin-conjugated anti-B220 (RA3-6B2), -IgM^a (DS-1), -CD24 (M1/69), -CD23 (B3B4), -CD138 (281–2), -CD19 (1D3) and -CD21 (7G6); allophycocyanin-conjugated anti-IFN-γ (XMG1.2); Alexa Fluor488-conjugated anti-IL17A (TC11-18H10); and PE-anti-IL21 (4A9). Biotinylated peanut agglutinin (PNA) was purchased from Sigma-Aldrich (St. Louis, MI) and biotinylated polyclonal rabbit anti-HEL Ab was purchased from Rockland (Gilbertsville, PA). Isotype controls were purchased from Caltag (Buckingham, UK). Allophycocyanin-, PE-, or PerCP-conjugated streptavidin (BD Biosciences) were used to reveal biotinylated Ab staining. Dead cells were excluded by staining with propidium iodide (PI, Sigma-Aldrich), 0.6 μg/ml. Stained cells were acquired and analyzed using a BD FACSCalibur and FACSDiva software, or using a BD LSRII flow cytometer and FlowJo software (TreeStar, San Carlos, CA).

For immunolabeling, the following monoclonal antibodies (mAbs) were used: phycoerythrin (PE)-conjugated anti-CD4 (clone OX-38), biotin-conjugated anti-CD45 (clone OX-1), biotin-conjugated anti-CD134 (clone OX-40), peridinin-chlorophyll-protein (PerCP)-conjugated anti-TCRαβ (clone R73), FITC-conjugated anti-IFN-γ (clone DB-1), and PE-conjugated anti-IL-17A (clone TC11-18H10). These mAbs, as well as PerCP-conjugated streptavidin, APC-conjugated streptavidin, and PE-conjugated donkey anti-rabbit F(ab)2, as second-step reagents, were obtained from BD Biosciences Pharmingen (Mountain View, CA, USA). FITC-conjugated anti-CD11b (clone ED8) was purchased from Serotec (Oxford, UK), APC-conjugated anti-CD4 (clone OX-38) from eBioscience (San Diego, CA, USA), and rabbit anti-CX3CR1 polyclonal Ab from Abcam (Cambridge, UK).

For Ki67 cell cycle assay, bone marrow cells were labeled using biotinylated lineage antibody cocktail in combination with PerCP-conjugated streptavidin (BD, 554064) and antibodies against Sca-1, c-Kit, CD150, and CD48 as described under FACS protocol above. Labeled cells were fixed and permeabilized using Fixation/Permeabilization solution (BD, 554714) at 4 °C for 20 min and washed with 1x BD Perm/Wash buffer (BD, 554714). Cells were next incubated in 1x BD Perm/Wash buffer containing AlexaFluor674-conjugated anti-Ki67 antibody (BioLegend, 652408, 16A8 clone; 1:100) or AlexaFluor674-conjugated anti-IgG2 antibody (BioLegend, 400526, RTK2758 clone; 1:100) at 4 °C overnight. The next day, cells were washed with 1X BD Perm/Wash buffer and resuspend in 2% FBS containing 1xPBS. DAPI solution (Thermo, 62248; 1:500) was used to stain DNA before FACS analysis.