α cd4 apc cy7

For flow cytometry analysis, isolated cells (from cNTs, lymph nodes, or spleens) were aliquoted at 500,000 cells per 5 ml tube, and pre-treated with Fc block (hybridoma clone 2.4G2) prior to cell staining. Staining was done in panels using the following antibodies: α-CD3-APC (clone 145-2C11), α-CD3-PE Cy7 (clone 53–7.3), α-CD8 PE Cy7 (clone 53–6.7), α-CD45 PE (clone 30-F11), α-CD19 (clone MB19-1), α-NK1.1 (clone PK136), α-CD11c (clone N418), α-F4/80 (clone BM8), and α-GR1 (clone RB6-8C5) (all from eBioScience); α-CD4 APC Cy7 (clone GK1.5; Biolegend); and α-CD45 Alexafluor 700 (clone 30-F11; BD Biosciences). Dead cells were excluded using 7-AAD (BD Biosciences). Antibodies to stain cells for each panel were added, mixed, and incubated on ice in the dark for 30 minutes. Cells were washed twice with 1× PBS +5% FBS and resuspended in 500 µl of 1× PBS +5% FBS. Stained cells were run on a BD FACS Canto II flow cytometer (BD Biosciences). Standard compensations were used for each tissue using FACSdiva software.

αCD2-biotin (clone: RPA-2.10), αCD14-biotin (clone: HCD14), αCD16-biotin (clone: 3G8), αCD19-biotin (clone: 561), αCD235a-biotin (clone: HIR2), αCD3-biotin (clone: OKT3a), αCD34-APC (clone: 561), αCD38-PE/Cy7 (clone: HIT2), αCD4-APC/Cy7 (clone: RPA-T4), αCD8a-Pacific Blue (clone: RPA-T8), αCD90-PerCP/Cy5.5 (clone: 5E10), αCD272-FITC (αBTLA; clone: MIH26), αIgG2-FITC (clone: MOPC-173), αCD200R-PE (clone: OX-108), αIgG1-PE (clone: MOPC-21), αCD160-PerCP/Cy5.5 (clone: BY55), αIgM-PerCP/Cy5.5 (clone: MM-30), αCD96-APC (clone: NK92.39), αIgG1-APC (MOPC-21), αCD28-PE/Cy7 (clone: CD28.2), αIgG1-PE/Cy7 (MOPC-21) and αStreptavidin-FITC were from BioLegend, USA. αCD45-PE (clone: HI30) was from eBioscience, USA. αIgG1-PE (clone: IS11-12E4.23.20) was from Miltenyi Biotec, Germany. Samples were analyzed on BD LSRFortessa™ (BD Biosciences, USA). Data was analyzed using FlowJo software v.10.7 (FlowJo, LLC, USA).