Cd45 30 f11

Antibodies directed against mouse CD31 (PECAM-1) (affinity purified SL-4), raised in rabbit and purified as described elsewhere ^{20 (link)}. Rat monoclonal antibodies against mouse CD31 [clone: MEC13.3] (Cat No. 550274), CD45 [30-F11] (Cat No. 550539), CD11b [M1/70] (Cat No. 550282), Mac3 [M3/84] (Cat No. 550292), and VE-cadherin (VEC) [11D4.1] (Cat No. 550548) were purchased from BD Biosciences (San Jose, CA, USA). Rabbit polyclonal antibodies against Ki67 (Cat No. ab15580) were purchased from Abcam (Cambridge, MA, USA). Rabbit monoclonal antibodies against Survivin [71G4B7] (Cat No. #2808), YAP [D24E4] (Cat No. #8418), and phospho-YAP (Ser127) [D9W2I] (Cat No. #13008) (P-YAP) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). A mouse monoclonal antibody against glucose transporter 1 (GLUT-1) [SPM498] (Cat No. ab40084) was purchased from Abcam (Cambridge, MA, USA). Mouse monoclonal antibodies against human CD31 [JC70A] (Cat No. M0823), a CD45 [2B11+PD7/26] (Cat No. M0701) and a monoclonal mouse anti human CD68 were purchased from DAKO. Rabbit polyclonal antibodies against human Ajuba (Cat No. HPA006171) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). A rabbit polyclonal against human GLUT1 was purchased from Cell Marque (Rocklin, CA).

Cells were released from culture plates using TrypLE and were stained after Fc-block (Invitrogen) with a panel of directly fluorochrome-conjugated mAbs against the following murine molecules (clone; source): Axl (R&D; Minneapolis, MN); CD4 (L3T4; eBioscience; San Diego, CA); CD8 (CD8b.2; Biolegend; San Diego, CA); CD11b (M1/70; eBioscience); CD11c (N418; eBioscience); CD19 (MCA1439F; AB Serotec); CD44 (IM7; eBioscience); CD45 (30-F11; BD); CD80 (16-10A1; BioLegend); CD206 (C068C2; BioLegend); Ly6C (AL-21; BD); Ly6G (1A8; BD, Franklin Lakes, NJ); Mertk (R&D). Experiments were performed on an LSR II flow cytometer and data were analyzed as previously described (25 (link)).

To determine ex vivo T cell functionality from tumor-bearing mice, single cell suspensions from spleen and tumor were obtained and activated in vitro^{30 (link)}. Briefly, mononuclear cells were restimulated with 1X Cell Stimulation Cocktail (eBioscience) in the presence of Golgiplug and Golgistop (BD) according to manufacturer’s instructions in T cell media. 4–5 hours later, cells were stained with live/dead ghost dye at 1:500 (Tonbo) and the following antibodies at diluted in FACs buffer at 1:200 against CD45 (30F-11, BD), CD3 (17A2, Biolegend), CD4 (RM4.5, Tonbo), CD8 (53–6.7, Tonbo), Klrg1 (2F1, eBioscience), and CD44 (IM7, Tonbo) for 30 minutes at 4°C in the dark. Cells were washed 2X in FACs buffer, fixed/permeabilized using the BD cytofix/cytoperm kit (BD) and stained with anti-IFNγ (XMG1.2, Biolegend) diluted 1:100 in perm/wash buffer for 1 h at 4°C. Cells were washed 2X in perm/wash buffer, resuspended in FACs buffer and stored overnight at 4°C in the dark. Cells were acquired the following day on a Fortessa 1770 flow cytometer following the addition of counting beads (Sigma) and analyzed using FlowJo software (version 10).