Anti mouse cd45 percp cy5

DC isolation from tissue specimens was performed as described previously (35 (link)). Briefly, LN and thymus were digested with collagenase D (120μg/ml) and DNAse I (120μg/ml) (both from Roche, Austria) in RPMI 1640 medium (Sigma Aldrich). Whole trunk skin was cut into small pieces and digested with Liberase TM (150μg/ml, Roche, Austria) and DNAse I in RPMI 1640 medium. Skin specimens were incubated for 45 min and LN and thymus specimens for 30 min at 37°C with agitation. Cells were filtered through 70 μm nylon mesh filters and washed with PBS. Cells were stained with anti-mouse CD45-PerCP/Cy5.5 (clone: 30-F11, BioLegend), anti-mouse CD11c-BV421 (clone: N418 BioLegend), anti-mouse EpCam-PeCy7 (clone G8.8, BioLegend), anti-huLangerin-A647 (clone: 808E10.01, Dendritics), live/dead-BV500 (eBioscience). Cells were washed and resuspended in PBS/10mM EDTA to prevent cell aggregation. Fluorescent stains and YFP expression in LCs was analyzed by flow cytometry on a FACS Canto II (Becton Dickenson).

50 μL peripheral blood or ~5×10⁵ bone marrow cells in PBS with 10% FBS were blocked with anti-mouse CD16/CD32 (cat # 101302, Biolegend, San Diego, CA, USA) and then stained with fluorophore conjugated antibodies. Following staining, red blood cells were lysed with RBC lysis buffer (cat # sc-296258, Santa Cruz Biotechnology, Dallas, TX, USA) for 10 min and washed twice with 10% FBS in PBS. Peripheral blood samples underwent a second 5 min RBC lysis prior to washing. Samples were acquired on a BD Accuri C6 or Beckman coulter Gallios flow cytometer and analyzed using BD Accuri Csampler software or FlowJo. Cells were stained with anti-human CD34 PE (cat # 343606, Biolegend), anti-human CD11b PE (cat # 555388, BD Biosciences, San Jose, CA, USA), anti-human CD3 PE/Dazzle™ 594 (cat # 300450, Biolegend), anti-mouse CD45 PerCP-Cy5.5 (cat # 103132 Biolegend), anti-mouse CD45 PerCP (cat # 557235, BD), anti-human CD19 PE-Cy7 (cat # 302216, Biolegend), anti-human CD45 APC (cat # 555485, BD Bioscience or cat # 304012, Biolegend). Analysis based 10 000 bone marrow and 3 000 blood cells from the live cell gate.

Primary murine HNSCC tissues and cervical lymph nodes were harvested from 4NQO-induced mice treated with vehicles or anti-CD276 antibodies for 10 days. Single cell suspensions were achieved as described above. For staining of cell surface markers, cells were incubated with indicated antibodies on ice for 30min and washed with staining buffer. Then, the stained cells were examined using a flow cytometer (Navios, Beckman Coulter) and the data was analyzed with CytExpert software (https://www.beckman.com/flow-cytometry/instruments/cytoflex/software). Antibodies used for flow cytometry analysis are listed below: anti-mouse FVS700 APC-700 (BD Biosciences, Cat#564997), anti-mouse CD45 Percp-cy5.5 (BioLegend, Cat#103132), anti-mouse CD4 PE-cy7 (BioLegend, Cat#100422), anti-mouse CD3 Alexa Flour488 (BioLegend, Cat#100210), anti-mouse CD8 BV786 (BD Biosciences, Cat#563332), anti-mouse NK1.1 BV510 (BioLegend, Cat#108738), anti-mouse Gr-1 PE/Dazzle 594 (BioLegend, Cat#108452), anti-mouse CD11b BV605 (BioLegend, Cat#101257), and anti-mouse F4/80 BV650 (BioLegend, Cat#123149).

Leukocytes: Blood was collected via the tail vein with EDTA-coated capillaries. Red blood cells were lysed with RBL buffer (Sigma Aldrich), and blocking achieved with antimouse CD16/CD32 (eBioscience). Monocytes were identified by staining with anti-mouse CD45-PerCp/Cy5.5 (Biolegend), anti-mouse CD115 (CSF-1R)-Brilliant Violet 605 (Biolegend) and antimouse Ly-6G/Ly-6C-APC (Biolegend). Monocyte activation was determined via by CD11b expression (geo-mean fluorescence intensity [MFI]). Neutrophils were identified as CD45^hiCD115^loLy6-C/G^hi. Monocyte-platelet aggregates were identified by first gating on monocyte population of interest and identifying those positive for platelets with CD41. Flow cytometry was performed using a LSRII analyzer. Whole blood counts were recorded on a CBC (Oxford Sciences). Platelets: Blood was collected via retro orbital bleeding with EDTA-coated capillaries, and diluted in heparin. Antibodies against P-selectin-AlexaFluor 647 (BD Biosciences) or JON/A-PE (Emfret) were added to whole blood, and platelet agonists added in accordance with the figure legend. Samples were incubated for 15 min, prior to dilution in Tyrode’s buffer, and immediately analyzed via flow cytometry (BD Accuri). Platelets were identified by forward scatter and side scatter characteristics.