Dulbecco phosphate buffered saline (dpbs)

To compare the binding of C3b to FH1–4_WT and FH1–4_W198R by ELISA, microtiter plate wells were coated with 100 nM of FH1–4_WT and FH1–4_W198R in Dulbecco’s PBS (DPBS; Lonza) at 20°C for 1 h. The wells were washed after each step with DPBS containing 0.05% Tween-20. After blocking with 5% BSA in DPBS-Tween for 1 h, serial dilution of C3b was added in DPBS at 20°C for 1 h. Bound C3b was detected with HRP-conjugated anti-C3 antibody, using 3,3′,5,5′-tetramethylbenzidine (TMB) (BioLegend; Biomedica, Budapest, Hungary) as chromogen and reading the absorbance at 450 nm.
The interaction of FH1–4_WT and FH1–4_W198R with C3b was also analyzed by surface plasmon resonance, using ProteOn XPR36 equipment (BioRad). Recombinant FH1–4_WT and FH1–4_W198R were diluted in acetate buffer (pH 4.0) and immobilized on two parallel flowcells on a GLC biosensor chip using a standard amine coupling protocol, as recommended by the manufacturer, to achieve a coupling density of ~3,500 RU for the two proteins. Serial dilutions of C3b were used as analytes, diluted in PBS containing 0.005% Tween-20 and 145 mM NaCl as running buffer at a flow rate of 30 µl/min. Association was followed for 300 s and the dissociation for 600 s. Data were analyzed with ProteOn manager software.

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).

IL-1β cytokine was measured from the islet supernatants using sandwich ELISA following the manufacturer’s protocol (n = 4). Briefly, Nunc-Immuno 96-well plates were coated overnight (at 4°C) with purified anti-human IL-1β antibody at 1μg/mL in DPBS (BioLegend, California, USA, Cat. No. 511601). The next day, plates were blocked with DPBS + 1% BSA for 2 hours at 25°C, and then samples and standards were added to the 96-well plate in 2 replicates and incubated for 2 hours at 25°C. A dilution of 1:2 was used for the samples with the standards ranging from 20 to 5000pg/mL (BioLegend, Cat. No. 579409). Plates were subsequently incubated with biotin-conjugated antibody at 2μg/mL for 1 hour at 25°C (BioLegend, Cat. No. 511703), followed by incubation with Streptavidin-alkaline phosphatase (1:2000 dilution, BD Biosciences, Cat. No. 554065) for 1 hour at 25°C. Plates were washed 3 times with 1xDPBS containing 0.05% Tween-20 after each incubation step. Color was developed by adding PNPP substrate (Sigma-Aldrich) and absorbance was detected at 405nm using SpectraMAX 250 spectrophotometer (Molecular Devices Corp., California, USA). The amount of IL-1β in each experimental sample was determined from the absorbance value plotted against the concentration of a standard curve for the cytokine.