Alexa fluor 647 conjugated anti mouse f4 80 antibody

Fresh tumor tissues were cut into small pieces and were incubated in a combination of 0.15% type I collagenase (Sigma-Aldrich; catalog no. C0130-500MG) and 0.15% type II collagenase (Sigma- Aldrich; catalog no. C6885-1G) at 37 °C for 1 h. Digested tissues were filtered with a 100-μm cell strainer, followed by a 70 μm cell strainer. Single cells from each sample after centrifugation were stained on ice for 15 min with an Alexa Fluor 647–conjugated anti-mouse F4/80 antibody (123122, BioLegend), an APC–conjugated anti-mouse CD3 antibody (17-0032-80, BioLegend), an APC–conjugated anti-mouse CD4 antibody (100411, BioLegend), and a PerCP–conjugated anti-mouse CD8 antibody (100731, BioLegend). The stained samples filtered with a 40-μm cell strainer were scanned on a FACScans (Becton Dickinson) and data were analyzed with a CellQuestPro software (BD Biosciences). The gating strategy of flow cytometry is presented in Supplementary Fig. 11.

Fresh tissues were cut into small pieces in ice-cold PBS, and then digested in PBS containing 0.1% collagenase I and II (catalog 40507ES60, YEASEN; catalog 40508ES60, YEASEN) in 37°C for 30 minutes with gentle pipetting. After digestion, cells were washed with ice-cold PBS and resuspended by 1 mL MACS buffer (a solution containing PBS, 0.5% BSA, and 2 mM EDTA) and stained with an Alexa Fluor 647–conjugated anti–mouse F4/80 antibody (catalog 12322, BioLegend) or a rabbit anti–mouse NG2 antibody (catalog AB5320, Millipore), followed by an Alexa Fluor 647–conjugated donkey anti-rabbit antibody (catalog A31573, Invitrogen). Anti–Alexa Fluor 647 MicroBeads (catalog 130-091-395, Miltenyi Biotec; catalog 130-042-303, Miltenyi Biotec) were subsequently used for magnetic labelling. After washing, positive and negative cells were sorted with a MACS column and magnetic MACS separators (catalog 130-042-201, Miltenyi Biotec). NG2⁺ and NG2^– populations were collected for following experiments.