Anti hamster

The following antibodies were purchased from BioLegend, eBioscience, or BD Biosciences and used for flow cytometry or enrichment: FITC-Sirpα, PE/Cy7-Sirpα, PerCP/Cy5.5-B220, APC-EpCAM, PE-CD11c, PE/Cy7-CD11c, Brilliant Violet 605-CD11c, Pacific Blue-IA/IE, FITC-CD8α, Alexa700-CD8α, PE-V_α2, APC-V_α2, APC-V_β5, PE-V_β6, PE-Foxp3, Pacific Blue-Foxp3, PerCP/Cy5.5-Thy1.1, APC-TCRβ, PE-CD69, PE/Cy7-CD69, Brilliant Violet 605-CD4, APC-CD25, Brilliant Violet 605-CD25, Brilliant Violet 785-CD25, FITC-CD45RB, APC-ICAM1, PR-CD9, PE-CD81, APC-CD48, PE/Cy7-CD24, APC-CD24, PE-CD71, PE/Cy7-CD45, APC-streptavidin, Biotin-Thy1.2, Biotin-CD8β, Biotin-CD25, and Biotin-B220. Alexa647-CTxB subunit was purchased from Molecular Probes. For Western blotting, the following clones were used: MHCII β chain (Bett; homemade), CD48 (OX-78; Serotec), CD24 (M1/69; BioLegend), actin (A2066; Sigma), CD9 (LMC8; eBioscience), and CD81 (Eat2; BioLegend), and HRP-conjugated secondary antibodies (anti-rat, anti-rabbit, and anti-hamster) were purchased from Jackson ImmunoResearch or BIOSOURCE. Myriocin, MβCD, cholesterol, sphingomyelin, and 4-hydroxy-tamoxifen were purchased from Sigma.

Forty-eight hours after transfection, mouse melanoma B16F1 were detached with nonenzymatic cell-dissociation solution (C5788; Sigma), washed twice in PBS containing 0.5 mg/mL BSA, and split into two equal populations. Antibody staining or soluble ligand incubation was performed in PBS containing 0.5 mg/mL BSA on ice. For one cell population, the total amount of cell-surface integrin was revealed by staining with a hamster anti–β3-integrin (anti-mouse CD61; Pharmingen, BD). The other cell population was incubated with an arginylglycylaspartic acid-containing fusion protein of the snake venom disintegrin (Kistrin) and the first Ig-domain of CD31 (SKI-7), followed by the incubation with a rat anti-CD31 monoclonal antibody (GC51) as described (25 (link)). Subsequent detection of the anti–β3-integrin and the bound β3-integrin ligand (SKI-7)/GC51 complex was achieved with phycoerythrin-labeled affinity purified anti-hamster (127.115.161; The Jackson Laboratory) or F(ab')₂ fragments of goat anti-rat antibodies, respectively, and analyzed in an Accuri flow cytometer. Integrin activation was determined by the ratio of the signal between the activation-specific and total integrin-binding antibodies as described in ref. 25 (link).