Anti cd206 pe cy7

Single‐cell suspension from tumors was analyzed by flow cytometry as described earlier [15 (link)]. Briefly, cells were incubated with Fc receptor blocker followed by staining with anti‐CD45‐ Brilliant Violate 421, anti‐F4/80 PerCP/Cy5.5, anti‐CD11b APC/Cy7, anti‐CD206 PE/Cy7, anti‐CCR7 Brilliant Violate 785, and anti‐Gr1 FITC (BioLegend, San Diego, CA, USA). For staining intracellular targets, cells were fixed and permeabilized for 30 min at room temperature (Fixation/Permeabilization Diluent, eBioscience, Waltham, MA, USA). Cells were stained with anti‐EpCAM‐APC (BioLegend). A total of 10 × 10⁶ cells were recorded on a flow cytometer. All the data was recorded on FACS Fortessa (BD Biosciences, Waltham, MA, USA) and analyzed using flowjo software (FlowJo LLC, Ashland, OR, USA).

Intracellular IFN-α staining was performed according to the intracellular staining protocol from BD Bioscience using anti-human IFN-α antibody APC (Miltenyi Biotec). Surface marker staining with anti-CD14 V450 (BD Bioscience), anti-CD163 PE, anti-CD206 PE-Cy7, and anti-CD209 APC (BioLegend) was performed for 20 min at 4°C. Data were acquired on a LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).

SVF was isolated from scWAT or eWAT and resuspended in red-blood-cell lysis buffer for 5 min to remove blood red cells. The collected cell pellet was incubated with LIVE/DEAD™ Fixable Violet Dead Cell Stain (Thermo Scientific, L34955, 1:1000) at 22 °C for 10 min for live-dead staining. After washing with PBS, the cell pellet was blocked by anti-CD16/32 (BioLegend, 1:1000) and then incubated with the following antibodies for 20 min at 4 °C: anit-CD45-APC Cy7 (BioLegend, 103116, 1:300), anti-F4/80-FITC (Cell Signaling, 52267, 1:100), anti-CD11c-AF647 (BioLegend, 117314, 1:100), anti-CD206-PE Cy7 (BioLegend, 141720, 1:100). After washing and resuspended with FACS buffer, cells were subjected to analyze by CytoFLEX S flow cytometer (Beckman Coulter) and sequential plot analysis was performed with the Flowjo software (v10).