Percp cy5.5 conjugated anti cd45

A CNS or spleen leukocyte suspension was obtained as described previously.^{49 (link)} Freshly isolated cells (10⁶) were incubated with an anti-mouse CD16/CD32 FC (eBioscience) and labeled with anti-mouse antibodies: PE anti-CD25; PE anti-CD3; PerCP-Cy5.5-conjugated anti-CD45; PerCP-Cy5.5-conjugated anti-B220; PE-Cy7-conjugated anti-CD39; PE-Cy7-conjugated anti-CD8a; PE-Cy7-conjugated anti-CD19; APC anti-CD3; APC-Cy7-conjugated anti-CD4; APC-Cy7-conjugated anti-CD11b (all from BD Pharmingen); APC anti-CD3; and PE-Cy7-conjugated anti-CD39 (both from eBioscience). To detect Foxp3, the cells were suspended in the fixation/permeabilization buffer and stained with a PE anti-Foxp3 antibody (BD Pharmingen). Cell viability was assessed with the LIVE/DEAD Fixable Green Dead Cell Stain Kit or LIVE/DEAD Fixable Red Dead Cell Stain Kit (Life Technologies Corporation). At least 10,000 events were acquired in each experiment on a FACSaria flow cytometer (BD Biosciences) and the data were analyzed using FlowJo software (v.10; Tree Star).

A CNS and spleen leukocyte suspension was obtained as described previously (Carrillo‐Salinas et al., 2017 (link)). Isolated cells were incubated with anti‐CD16/CD32 (Affymetrix Inc.) for FcR blockade and labeled with anti‐mouse antibodies: PE‐conjugated anti‐CD44 (2.4 μg/ml), PE‐conjugated anti‐CD274 (2.4 μg/ml), PerCP‐Cy5.5‐conjugated anti‐CD4 (1.2 μg/ml), PECy7‐conjugated anti‐Ly6c (1.2 μg/ml), APC‐Cy7‐conjugated anti‐CD11b (1.2 μg/ml), APC‐conjugated anti‐CD62L (2.4 μg/ml), APC‐conjugated anti‐CD25 (2.5 μg/ml), and APC‐conjugated anti‐CD1d (2.4 μg/ml; all from eBioscience); APC‐conjugated anti‐P2yR12 (2.5 μg/ml) and APC‐Cy7‐conjugated anti‐CD3 (1.2 μg/ml) (both from Biolegend); PE‐conjugated anti‐CD5 (2.2 μg/ml), PerCP‐Cy5.5‐conjugated anti‐B220 (2.4 μg/ml), PerCP‐Cy5.5‐conjugated anti‐CD45 (1.2 μg/ml), PECy7‐conjugated anti‐CD8 (1.2 μg/ml) and PECy7‐conjugated anti‐CD19 (2.4 μg/ml; all from BD Pharmingen). The cells were fixed for 30 min with fixation buffer (Affymetrix Inc.). For FoxP3 detection, the cells were suspended in Fixation/Permeabilization buffer for 30 mins prior to staining with an anti‐FoxP3 antibody (3 μg/ml; BD Pharmingen). At least 50,000 events were registered in each experiment on a FACSAria flow cytometer (BD Biosciences), excluding duplets from the analysis. The data were analyzed using FACSDiva analysis software (BD Biosciences).

Cells were preincubated 10 min with anti-CD16/32 FcγII/III (BD Bioscience) to prevent nonspecific binding, washed and stained for surface markers, fixed in Fix/Perm buffer (BD Bioscience), and permeabilized in permeabilization buffer (BD Bioscience) for 1 h with antibodies. For intracellular staining, GolgiStop (BD Bioscience) was added 6 h before Fc block, fixation, and permeabilization (BD Bioscience). Primary antibodies for flow cytometry were: PerCP/Cy5.5-conjugated anti-CD45 (BD Biosciences), APC-conjugated anti-CD11b (BD Bioscience), PE-conjugated anti-CD117/c-Kit (BioLegend), FITC-conjugated anti-FcεRIα (BioLegend), PE/Cy7-conjugated anti-T1/ST2 (IL-33R; eBioscience), APC-conjugated anti-IL-22 (eBioscience), and PE/Cy7-conjugated anti-IL-13 (eBioscience). Acquisition was with a BD FACSCanto instrument. Data analyses used FlowJo software (Tree Star).