Anti cd4 percp cy5

After isolation by Percoll gradient for adoptive transfer (see above), sets of unlabeled lymphocytes were incubated (30 min, 4°C) with antibodies, as follows: (i) anti-mouse CD8a FITC (0.5 μg, eBioscience, Cat. code: 11-0081); (ii) anti-CD4 PerCP-Cy5.5 (0.5 μg, eBioscience, Cat. code: 45-0042-82); (iii) anti-TCRβ PE-Cy7 (0.5 μg, eBioscience, Cat. code: 25-5961-82), and (iv) anti-CD19 FITC (0.5μg, BD, Cat. code: 553785). Cells were washed and 50,000 events were acquired from each sample in a cytometer (Gallios, Beckman Coulter) and analyzed with FlowJo 10.0.5 software (Tree Star Inc., Ashland, OR, USA).

DCs were harvest after 24 h treatment and stained with different antibodies raised against specific surface markers: Anti-CD11C- PerCP-Cy5.5, Anti-CD86-APC, Anti-CD40-FITC, Anti-CD80-PE-Cy7, Anti-MHCII-PE, Anti-CD274 (PD-L1)-PE, Anti-CD273(PD-L2)-FITC, Anti-OX40-L-APC for 30 mins. Co-cultured cells were harvest and then mixed with different antibodies raised against specific surface markers: Anti-CD4-PerCP-Cy5.5 (eBioscience, The Netherlands), anti-CD25-FITC (eBioscience, The Netherlands), anti-CD69-PE (eBioscience, The Netherlands), anti-CCR-6-PE (eBioscience, The Netherlands), for 30 minutes. Staining for intracellular markers were performed according manufacturer’s protocol, (eBioscience, Foxp3 staining set, Bio connect, The Netherlands). Antibodies used for intracellular markers where anti-Foxp3-PE-Cy7 (eBioscience, the Netherlands), anti-RORγt-PE (eBioscience, The Netherlands). Matching Isotype controls were used to minimize the influence of nonspecific binding and proper gate setting. All incubations were performed on ice and protected from light. In total a minimum of 50,000 cells were counted and analyzed using FACS Canto II and FACS Diva software (BD Biosciences, The Netherlands).

Colons were cut into ~1 cm² pieces, washed in 1X Hank’s Balanced Salt Solution (HBSS) containing 2% heat-inactivated fetal bovine serum (FBS), disrupted by vortexing, further treated with collagenase IV (20 mg/ml) and DNase I (10 mg/mL) in RPMI medium and passed through a 70 μM cell strainer to collect lamina propria cells. Cells were treated with blocking antibodies to CD16/32 (1:100), and stained for viability using Zombie Viability Dye V500 (1:400). Surface staining was done on ice for 20 min using anti-CD45 APC-eFluor780 (1:400, clone 30F11, eBioscience), anti-CD11b APC (1:400, clone M1/70, InVitrogen), anti-Gr1 PerCp-Cy5.5 (1:300, clone RB6-8C5, Biolegend), anti-Siglec F PE (1:400, clone E50-2440, BD), anti-CD3 FITC (1:200, clone 145-2C11, Biolegend), anti-CD49b FITC (1:200, clone DX5, eBioscience), anti-B220 FITC (1:300, clone RA3-6B2, eBioscience), anti-CD4 PerCp-Cy5.5 (1:400, clone RM4-5, eBioscience), and anti-CD8a AlexaFluor 700 (1:200, clone 53–6.7, Biolegend),. Intestinal epithelial cells were collected as described in^{22 (link)}. Cells were stained with anti-EpCAM PE-Cy7 (1:400, clone G8.8, eBioscience) and anti-FITC CD44 (1:200, clone IM7, eBioscience). Cells were analyzed on the Fortessa (BD Biosciences) and data analysis was performed on FlowJo (Tree Star).