Anti mouse cd11b percp cy5

Tumor tissues were resected from mice and minced into small pieces and then were lysed by 1 mg/ml collagenase IV (Sigma, United States) and DNase I (Invitrogen, United States) for 1 h at 37°C. Afterward, the tissue medium was filtrated using a 70-μm filter screen to obtain single-cell suspensions. The cell suspensions were stained with antibodies for 30 min and washed three times by PBS and then were subjected to FCM analysis. The following reagents and antibodies were used in FCM analysis.
Panel A: LIVE/DEAD™ Fixable Stain (Invitrogen, California, United States), anti-mouse CD45-BV605 (Biolegend, California, United States), anti-mouse CD3-PE-cy7 (Biolegend, California, United States), anti-mouse CD4-Efluor450 (BD, New Jersey, United States), anti-mouse CD8-Percp-cy5.5 (Biolegend, California, United States), anti-mouse CD19-BV650 (Biolegend, California, United States), and anti-mouse NK1.1-PE (Biolegend, California, United States).
Panel B: LIVE/DEAD™ Fixable Stain (Invitrogen, California, United States), anti-mouse CD45-BV605 (Biolegend, California, United States), anti-mouse CD11b-Percp-cy5.5 (Biolegend, California, United States), anti-mouse F4/80-PE (Biolegend, California, United States), and anti-mouse Ly6G-APC (Biolegend, California, United States).

Murine pre- and post-enrichment bone marrow cells were incubated with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain (Thermo Fisher Scientific Inc., #L34975) and the anti-CD45-FITC (1:100, BioLegend, #103107), anti-mouse c-kit-APC (1:100, BioLegend, #105811) and anti-mouse CD11b-PErCP/Cy5.5 (1:100, BioLegend, #101227) primary antibodies. Samples were then analyzed in a NovoCyte flow cytometer (ACEA Biosciences Inc.). For zebrafish chimera cell analyses at 2 dpf, embryos were euthanized with tricaine in E3 medium (8-12 embryos per condition) and mechanically dissociated by sterile razor blades or 0.05% trypsin (Gibco™). A single-cell suspension was prepared by collecting dissociated tissue in 0.5 ml 0.9× PBS/2% FBS and passing the sample through a 40-µM nylon mesh. Samples were subjected to 3 nM DRAQ-7 Dead Cell Stain (Abcam, #ab109202) or LIVE/DEAD™ Fixable Near-IR Dead Cell Stain and incubated with the aforementioned antibodies or Ter119-APC (1:100, BioLegend, #116211), CD19-APC (1:100, BioLegend, #152409), CD3-APC (1:100, BioLegend, #100235), Gr1-APC (1:100, BioLegend, #108411), F4/80-APC (1:100, BioLegend, #123115) or CD11b-PE (1:100, BioLegend, #101207) and analyzed by flow cytometry on a BD FACSAria II (Becton Dickinson) or a NovoCyte flow cytometer. Data analysis was performed in FlowJo v10.

Migration of dendritic cells to LN was evaluated following a contact sensitization assay 16 (link). Briefly, the animals were sacrificed 18 h after an epicutaneous application of a solution containing fluorescein isothyiocyanate (FITC) (Sigma Aldrich cat. #F7250-250MG), dibutyl phtalate and acetone (Sigma Aldrich), and corresponding skin-draining LNs were collected and enzymatically digested in collagenase D (Cedarlane cat. #11088882001) for 25 min at 37 °C. Cells were passed through a 70 µm strainer, washed, counted and stained for analysis by flow cytometry (BD FACSCelesta^TM). The following fluorescence-conjugated antibodies were used: anti-mouse CD11b PerCp-Cy5.5 (BioLegend cat. #101228), anti-mouse CD11c BV786 (BD Biosciences cat. #563735) and anti-mouse MHCII PE (Tonbo Biosciences cat. #35-5321-U100). The percentage of FITC-positive dendritic cells that migrated to the corresponding draining lymph nodes was analyzed with FlowJo™ software (Tree Star Inc.).

Acriflavine (A8251), Rapamycin (SML 1657), EGF (Sigma RP 3027), p-EGFR (CST 2234S), p-S6 (CST 4858S), ATP (Sigma A2383), Suramin (Sigma S2671), 2-DG (Sigma D8375), Gefitinib, PMA were obtained from Sigma Aldrich (US). Reagents for ELISA, including anti-mouse IFNγ, anti-mouse TNFα, anti-mouse IL-17A, anti-mouse IL-10 (purified and biotinylated) were obtained from Biolegend (California). Western blot antibodies, anti-human HIF1α, anti-human mTOR, anti-human p70-S6, and anti-human β-actin were purchased from CST (US). Purified anti-mouse HIF1α antibody for immunohistochemistry was purchased from R&D systems (US). Anti-mouse CD3 BV510, anti-mouse γδTCR FITC, anti-mouse Gr1 BV421, anti-mouse CD11b PerCp-Cy5.5 for immunophenotyping were purchased from BioLegend (California). Gentamycin, SS agar (Hi media M108D), LB media, RPMI-1640 (Sigma AL060A; 500ml), L-glutamine (Invitrogen A2916801), Pen/Strep (Thermo Scientific 11360070), Tween 20 (Sigma 9005-64-5), ENZO compound library.

1 × 10⁶ single suspension cells were stained with the proper antibodies diluted in cell staining buffer (420201, BioLegend, USA) for 30 min at 4 °C. For surface staining, anti-mouse CD45-Alexa Fluor^® 700 (Biolegend, 109821), anti-mouse CD11b-PercpCy5.5 (Biolegend, 101227), anti-mouse CD3-PE (Biolegend, 100206), anti-mouse CD3-FITC (Biolegend, 100203), anti-mouse CD4-APC (Biolegend, 100412), anti-mouse CD8-PercpCy5.5 (Biolegend, 100721) and anti-mouse NK1.1-APC (eBioscience, 17-55941-82) were used for extracellular staining. For intracellular staining, the cells were stimulated with or without various cytokines and GolgiPlug for 4 h and then fixed/permeabilized with BD Cytofix/Cytoperm kit as the recommendation of the manufacturer. After washing with wash buffer, anti-human IFN-γ-Percp-cy5.5 (Invitrogen, 45-7319-42), anti-human and Granzyme B-PE (Invitrogen, 12-8899-41) were used for intracellular staining. All samples were determined using FACSalibur flow cytometer (Becton-Dickinson, FL, NJ, USA), and the percentages of cells within each phase of the cell cycle were analyzed using FACS Express 7 software (De Novo Software).