Live dead marker zombie uv

Cells collected in suspension (CAFs or neutrophils) were stained with a live/dead marker Zombie UV (1:1000, Biolegend) for 30 min at room temperature in DPBS (Gibco). Cells were then washed and stained with an anti-FAP-APC antibody (1:20, R&D Systems) for 20 mins at 4°C in DPBS supplemented with 2% FCS. After washing cells were fixed in a 1:1 solution of fixation buffer (Biolegend) and DPBS with 2% FCS overnight at 4°C before data acquisition on a LSR6Fortessa analyser (BD Biosciences). Flow cytometry data was then analysed using FlowJo version 10.7.1. Compensation was carried out using single stain control UltraComp eBeads (Invitrogen) and isotype control samples were stained using iso-anti-FAP-APC (1:20, R&D Systems). FAP expression was determined by gating on singlet, live cells and then looking at anti-FAP-APC signal compared to the isotype control.

Following tissue digest, cells were resuspended in DPBS (Gibco) for staining by flow cytometry, with 1 million cells per condition. For all conditions other than the unstained control, cells were stained with a live/dead marker Zombie UV (1:1000, Biolegend) for 30 min at room temperature in DPBS (Gibco). Cells were then washed (centrifuged at 300 × g for 5 min) in DPBS supplemented with 2% FBS (FACs buffer) and incubated with FC blocker (Biolegend) for 10 min and then stained with surface marker antibodies (EpCAM, CD45, CD31, FAP, CD29, Podoplanin and PDGFRβ, see Supplementary Table S1 for details) or the corresponding isotype control antibodies for 20 min at 4 °C in FACs buffer. After washing cells were fixed with Cytofix fixation buffer (BD Biosciences) for 20 min at 4 °C. Cells were then washed in Perm/Wash buffer (BD Biosciences) and centrifuged at 300 × g for 5 min. Intracellular antibodies (αSMA and FSP-1) or the corresponding isotype controls were diluted in Perm/Wash buffer then added to cells and incubated in the dark for 30 min at 4 °C. After washing, cells were stored in DPBS with 2% FBS overnight at 4 °C before data acquisition on a LSR6Fortessa analyser (BD Biosciences). Compensation was carried out using single stain control UltraComp eBeads (Invitrogen).