Cd16 cd32 93

Solid tumors or lungs/tumors were collected for flow cytometric analysis. Solid subcutaneous tumors or lungs containing experimental pulmonary metastasis were diced to ~1–2 mm³ pieces and further digested to single cell suspensions by 1 h incubation at 37°C in DMEM with 10% FBS, 50 U/ml DNAse I and 250 U/mL Collagenase I (Worthington Biochemical Corporation, Lakewood NJ). Red blood cells were lysed with RBC Lysis Buffer (eBioscience).
Antibodies against the following proteins were purchased from BD Biosciences (San Jose, CA): CD45 (Clone 30-F11); BioLegend (San Diego, CA): CD3e (145-2C11), CD4 (RM4-5), CD8a (53–6.7), CD223 (LAG-3, C9B7W), CD274 (PD-L1, 10F.9G2), CD279 (PD-1, 29F.1A12), or eBioscience (San Diego, CA): CD16/CD32 (93). Cells were blocked with anti-CD16/CD32, and stained for surface markers according to standard protocols. All samples were acquired on the BD LSRII or Fortessa and analyzed using FlowJo version 9.6.2 (TreeStar Inc., Ashland, OR).

C57BL/6J mice were vaccinated intradermally at the right flank with either Alexa Fluor 647-labeled OVA (OVA-647, 10 µg) alone or OVA-647 (10 µg) + heat-iMVA (an equivalent of 10⁷ pfu) in a volume 100 µl PBS. Skin draining lymph nodes (dLNs) at the right inguinal area were harvested at 24 h post injection, digested with Collagenase D (400 U/ml, Roche Diagnostics) and DNase I (50 μg/ml, Roche Diagnostics), and analyzed by flow cytometry for OVA-647 intensities and CD86 expression of the migratory DC and resident DC populations in the skin dLNs. The antigens and clone designations for the antibodies are as follows: BioLegend: CD11c (N418, cat# 117320), CD11b (M1/70, cat# 101226), MHC-II (M5/114.15.2, cat# 107645), CD3e (145-2C11, cat# 100341), CD8a (53-6.7, cat# 140418); BD Biosciences: CD19 (1D3, cat# 562701), CD49b (DX5, cat# 563063), Thermo Fisher: CD16/CD32 (93, cat# 13-0161-82), CD103 (2E7, cat# 11-1031-82), CD207 (eBioL31, cat# 12-2075-82), and TER-119 (TER-119, cat# 48-5921-82). All antibodies were used at 1:200. Cells were analyzed on the BD LSR Fortessa or LSR II flow cytometer and data were analyzed with FlowJo software (version 10.5.3).

The cell suspension was taken 100 μL cells to a 1.5 mL centrifuge at a concentration of 1 × 106 mL‐1. Each tube was added with monoclonal antibodies specifically binding to different macrophage and lymphocyte subsets according to the detection requirements and incubated for 30 min at room temperature in the dark. Finally, the proportion of cells was collected and determined by flow cytometry (A00‐1‐1102, Beckman, USA). The following monoclonal antibodies were employed in the experiment: F4/80 (BM8, 11‐4801‐82), CD16/CD32 (93, 17‐0161‐82), CD206 (685641, MA5‐23594), CD3e (145‐2C11, MA5‐17658), CD4 (GK1.5, 12‐0041‐82), CD8a (53‐6.7, 17‐0081‐82), CD25 (PC61.5, 17‐0081‐82), and FOXP3 (FJK‐16s, 17‐5773‐82), which all obtained from the USA Thermo Fisher Scientific Co. Ltd.