Anti cd86 bv421

Upon cisplatin and CM treatment, macrophages were harvested using accutase (Sigma Aldrich, #A6964) and subsequent gentle scrapping of the culture wells. The harvested macrophages were washed once with PBS 2 mM EDTA and stained for 20 min with the following human antibodies: anti-HLA-DR-PE-Cy7 (Clone: L243, BioLegend, San Diego, CA, USA, #307616), anti-CD86-BV421 (Clone: IT2.2, BioLegend, #305426), anti-CD206-PE (Clone:19.2, BD Biosciences, Franklin Lakes, NJ, USA, #555954), and anti-CD163-BV510 (Clone: GHI/61, BioLegend, #333628). All antibodies were properly titrated before. Consecutively, the cells were also stained with the viability dye Sytox Bue (Invitrogen, #S34857). The cells were then acquired using flow cytometry (FACS Canto II, BD Biosciences) and the obtained .fcs data were analyzed with FlowJo 10 software (purity check, Supplementary Figure S2; changes in macrophage marker expression, Supplementary Figure S3).

Tumor-draining lymph nodes were mechanically passed through a 200-mesh filter. Nonspecific labeling was blocked with anti-mouse CD16/32 (Fc block, BioLegend, USA). DC maturation was examined by staining with Fixable Viability Dye eFluo 780 (eBioscience, USA), anti-CD45-Percy-cy5, anti-CD11c-APC, anti-CD80-PE, anti-CD86-BV421, and anti-MHCII-FITC antibodies, or an isotype IgG control (BioLegend, USA) [31 (link)]. For each sample at least 10⁴ cells were detected on a BD FACS Verse Flow Cytometer (BD, USA). Data obtained were analyzed using FlowJo 7.6 software (Tree Star, USA).