Anti cd68 alexafluor 488

Antibodies used for immunofluorescence were: anti-NAPRT1 (NBP1-87243, 1:100 Novus Biological) anti-p65 (sc-8008, 1:100 from Santa Cruz Biotechnology), anti-TLR4 (NB-10056566, 1:100 from Novus Biologicals), anti-CD68 AlexaFluor-488 (333812, 5 µl for well from Biolegend, San Diego, CA), anti-CD11b (HPA002274, 1:200 from Sigma-Aldrich, Saint Louis, MO). Secondary reagents were: goat anti-mouse IgG AlexaFluor488-conjugated (1:50) and goat anti-rabbit IgG AlexaFluor488-conjugated (1:100, both from Thermo-Fisher, Waltham, MA). After the primary and secondary antibodies, cells were counter-stained with AlexaFluor 568-conjugated phalloidin (1:100) and 4′,6-Diamidino-2-phenylindole (DAPI, 1:30,000, both from Thermo-Fisher).

Red blood cells were lysed using ammonium chloride solution according to manufacturer’s instructions (Qiagen, Cat# 1,045,722) for 10 min at room temperature prior to antibody staining. Tumour and tissue samples were digested using Type II collagenase (Sigma; Cat#C6885-100MG;) for three hours at 37 °C, washed and re-suspended in RPMI + 10% FBS; R10% (RPMI-1640 (Sigma-Aldrich; Cat# R8758) with 10% heat-inactivated fetal bovine serum, glutamine (1X), sodium pyruvate (1X) and Penicillin–Streptomycin (RPMI 10% = R10%). Immune populations were identified by staining with anti-human antibodies: anti-CD33-APC (BioLegend Cat# 303,408, RRID:AB_314352), anti-HLA-DR-PE (BD Biosciences Cat# 555,812, RRID:AB_396146), anti-CD14-PE-Cyanine7 (Thermo Fisher Scientific Cat# 25-0149-42, RRID:AB_1582276), anti-CD68-Alexa Fluor 488 (BioLegend Cat# 333,812, RRID:AB_2074832), anti-CD163-Brilliant Violet 421 (BioLegend Cat# 333,612, RRID:AB_2562463), anti-CD206-APC/Cyanine7 (BioLegend Cat# 321,120, RRID:AB_2144930) and anti-CD15-FITC (BioLegend Cat# 301,904, RRID:AB_314196) antibodies on ice for 30 min in Flow Buffer consisting of 1X PBS (Sigma-Aldrich: P3813) and 2%BSA (Sigma- Aldrich: 9048-46-8). Cells were acquired using a Cyan and CytoFLEX flow cytometers and analysed using FlowJo and CytoExpert2.2.