Recombinant murine cxcl9

CD8⁺ T cells were isolated from the spleens of wild type (WT) C57BL/6 mice using the mouse CD8a⁺ T-Cell Isolation Kit (Miltenyi Biotec, Germany). Cells were cultured with RPMI 1640 supplemented with 10% FBS, 1% P/S, and 100 U/ml recombinant murine IL-2. To perform a chemotaxis assay, 24-well Transwell chambers with 5-μm polycarbonate membranes were used. Recombinant murine CXCL9 (100 ng/ml, PeproTech, USA), CXCL10 (50 ng/ml, PeproTech, USA), and culture supernatants from transfected cells after 48 hours of culture were added to the bottom wells. Then, 1 × 10⁵ CD8⁺ T cells were seeded in the top chamber. After incubation for 2 h, the number of migrating cells was assessed using a hemocytometer.

Recombinant murine CXCL9 was purchased from Peprotech (USA). Selective STAT3 inhibitor cryptotanshinone was purchased from Selleckchem (USA). Anti-CD3ε and anti-CD28 antibodies were purchased from Biolegend (USA). Antibodies against phosphor-STAT3, STAT3, and β-actin were purchased from Cell Signalling Technology (USA). STATTIC was purchased from Sigma-Aldrich (USA).

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).