Cd3 pacific blue

To determine the absolute number of immune cells per µl whole blood, TRUcount™ tubes (BD Biosciences (BD), San José, California, USA) were used according to manufacturers’ protocol. In brief, 50 µl of whole blood was incubated with CD45-PerCP (BD), CD3-Pacific Blue (eBioscience Inc., San Diego, California, USA), CD8-APC-Cy7 (BD), CD4-PE-Cy7, CD16-PE, CD19-FITC and CD56-APC. Thereafter erythrocytes were lysed (BD lysisbuffer) and samples were measured on LSR-II FACS machine. At least 2000 lymphocytes were measured (identified as CD45⁺SSC^low) and analyzed with FACSdiva software (BD). We gated on CD3⁺CD4⁺ T-lymphocytes, CD3⁺CD8⁺ T-lymphocytes, CD3⁻CD16⁺ NK cells, CD3⁻CD56⁺ NK cells, and CD19⁺ B cells.

The following reagents were from BD Biosciences (San Jose, CA, USA) or eBiosciences (San Diego, CA, USA): Monoclonal antibodies to CD11b-FITC (M1/70), F4/80-PerCP5.5, CD11c-APC, CD3-pacific blue, CD4-FITC, CD8-APC, CXCL9-PE, CXCR3/CD183-PE and their corresponding isotype antibodies (Rat IgG2a-FITC, Rat IgG2a-PerCP5.5, Armenian hamster IgG-APC, Rat IgG2a-pacific blue, Rat IgG2a-FITC, Rat IgG2a APC, Hamster IgG-PE). Recombinant murine CXCL9 from Pepro Tech (Cranbury, NJ, USA) and purified mouse anti-CXCR3 antibodies (catalog number-155902, clone-S18001A, and lot number-B265189) was from Biolegend (San Diego, CA, USA). Liberace Cl was from Roche (Indianapolis, IN, USA). Bovine serum albumin (BSA), Gey’s balanced salt solution (GBSS), and DNase were from Sigma (St. Louis, MO, USA). Anti-CD11b, CD11c, CD4, and CD8 microbeads were from Miltenyi Biotec (Auburn, CA, USA). Diff-Quik stain set was from Dade Behring, Inc. (Newark, NJ, USA). Polycarbonate membranes, cell scraper, and Boyden chemotaxis chamber were from Neuro Probe, Inc. (Gaithersburg, MD, USA). ELISA kit for the detection of mouse CXCL9 was from R&D System (Minneapolis, MN, USA). LSRII flow cytometer from BD Biosciences (San Jose, CA, USA), FCS Express software from De Novo Software (Los Angeles, CA, USA).

For analysis of in vivo experiments, cells were stained with antibodies against human CD45 APC (Invitrogen), CD4 ECD (Beckman Coulter), CD3 Pacific Blue (eBioscience) CD8 PE and CD25 PE-Cy7 (BD) and the viability dye 7-AAD (eBioscience). To analyse expression of Treg-associated markers, freshly sorted or expanded Tregs were stimulated for 15h with anti-CD3/anti-CD28 beads at a ratio of 1 bead to 5 cells, or left untreated. Cells were stained with 7-AAD and antibodies against GITR FITC (R&D Systems), CTLA-4 PE, CD69 APC-Cy7, CD25 PE-Cy7 (BD), TIGIT PE, OX-40 FITC, TIM-3 APC, CD39 PE, FOXP3 eFluor 450, Perforin APC (eBioscience) Helios AlexaFluor 647 (Biolegend), and CD4 ECD (Beckman Coulter). For intracellular antigens (FOXP3, Helios, Perforin, CTLA-4), cells were fixed and permeabilized using a Foxp3 Staining Buffer Set (eBioscience). Samples were acquired using a BD FACSCanto (BD Biosciences) and analyzed using FACSDiva software (BD Biosciences). For staining for BCL-XL and MCL1, Abcam anti-BCL-XL FITC (7B2.5; ab26148) and anti-MCL-1 Alexa Fluor 488 (Y37, ab197529) antibodies were used, respectively, following manufacturer's instructions. Briefly, the cells were fixed with 4% paraformaldehyde and permeablized with PBS/0.1% Tween. The cells were then blocked with 10% normal goat serum/0.3 M glycine, followed by incubation with the antibody.