Streptavidin apc efluor 780

The following antibodies were used for flow cytometry: Anti-CD150-PE (TC15–12F12.2; 115904), anti-CD117-PE/Cy7 (2B8; 105814), anti-CD45.2-PE (104; 109807), anti-CD48-Alexa Fluor 647 (HM48–1; 103416) were purchased from BioLegend (San Diego, CA). anti-CD45-APC-eFluor 780 (30-F11; 47–0451-82), anti-Ter119-APC-eFluor 780 (TER-119; 47–5921-82), Streptavidin APC-eFluor 780 (47–4317-82), anti-CD31-PE/Cy7 (MEC13.3; 25–0311-82), anti-CD45.1-FITC (A20; 11–0453-85), anti-B220-APC-eFluor 780 (RA3–6B2; 47–0452-82), anti-CD4-PE/Cy7 (GK1.5; 25–0041-82), anti-CD8a-PE/Cy7 (53–6.7; 25–0081-85), CD51-PE (RMV-7; 13–0512-85), anti-PDGFRα (CD140a)-APC (APA5; 17–1401-81), anti-Ly6A/E(Sca-1)-FITC (D7; 11–5981-82), Anti-CD45-PerCp-Cyanine5.5 (30-F11; 45–0451-82), anti-CD11b (Mac-1)-PE (M1/70; 12–0112-83) were purchased from eBioscience (Thermo Fisher). Anti-lineage panel cocktail (TER-119, RB6–8C5, RA3–6B2, M1/70, 145–2C11 at 1:50 dilution) was from BD Biosciences (559971). Unless otherwise specified, all antibodies were used at a 1:100 dilution. All antibodies were anti-mouse and their specificity was validated in previous studies performed in our laboratory^{3 (link),8 (link),12 (link),27 (link)–29 (link)}.

The following antibodies against mouse antigens and conjugated to FITC, PE, PE-Cy5, PerCP-Cy5.5 or APC were purchased from BD Biosciences (CD4 (L3T4), eBioscience: biotin-rat anti-mouse CXCR5 (2G8), plus streptavidin-APC-eFluor 780 or eBioscience: PD-1 (J43), ICOS (7E17G9), Bcl-6 (mG1191E), IL-21 (mhalx21), CD8 (53–6.7), CD3ε (145–2C11), CD62L (MEL-14) and isotype controls.

Flow cytometry was performed on cells isolated from mice, as described previously [36 (link)]. Liver lobes were cut up and digested in 2.5 mg/ml collagenase (Roche, 11088866001) in 3 % FBS for 45 minutes at 37 °C. Tissues were cut up and mashed through 70 μm nylon filters in 10 ml of PBS. Cells were spun down, then red blood cell lysis was performed using Pharm Lyse (BD Biosciences, 555899) at 4 °C for 5 minutes, then quenched with PBS. For liver, blood cells were separated from the rest of the tissue using a 33 % percoll (GE Life Sciences, 17089102) separation spun at 800 g for 30 minutes. Cells were blocked for 30 minutes in MACS buffer (0.5 % FBS, 200 μM EDTA) and stained with the following antibodies: CD45-FITC (Biolegend, 147710), CD11b-BV605 (BD Horizon, 563015), Ly6G-AF647 (Biolegend, 127610), F4/80-BV711 (BD Horizon, 565612), Ly6C-biotin (Biolegend, 128003), streptavidin-APCefluor780 (eBioscience, 47-4317082), CD22-PE (Biolegend, 126111), CD19-APCCy7 (BD Pharmingen, 557655). T cells were stained using CD44-FITC, CD62L-PE, CD25-PE-Cy7, CD8-APC, and CD4-BV421. Flow cytometry was run on the Attune NxT in the Cytometry and Imaging Microscopy Shared Resource of the Case Comprehensive Cancer Center. Data was analyzed using FlowJo.