Isotype matched antibodies

Fibrocytes were identified using primary antibodies (all diluted 1∶20 in Hank’s balanced salt solution (HBSS), PAA, UK) against CD45RO (conjugated with phycoerythrin, PE, [BD Bioscience, UK]), 25F9 (conjugated with Alexa Fluor 647, AF647, [eBioscience, UK]) and MRP8/14 (conjugated with fluorescein isothiocyanate, FITC, [eBioscience, UK]). Samples were washed once with HBSS and centrifuged down into a pellet. Samples were then incubated in 100 µl diluted primary antibody cocktail on ice for 30 minutes. Cells were washed once with HBSS and resuspended in the same buffer in 500 µl HBSS for FACS analysis. 7AAD (BD Bioscience, UK) was added to determine cell viability. To assess non-specific binding, isotype-matched antibodies (eBioscience, UK) conjugated to corresponding fluorophores used in primary antibodies were used. Data were acquired on Accuri C6 (BD Bioscience, USA).

Flow cytometry was performed using a FACSCanto II or FACSAria III and analysed using FlowJo software (TreeStar). Dead cells were excluded with 7-AAD staining. Non-specific antibody binding was blocked with anti-CD16/32 antibody. Fluorescence-conjugated mAb against CD11b (M1/70), CD11c (N418), Gr-1 (RB6-8C5), F4/80 (BM8), Ly6C (AL-21), MHC class II I-Ab (AF6-120.1), CD103 (2E7), CD45 (30-F11), CD3 (145-2C11), CD4 (GK1.5), NKp46 (29A1.4), Thy-1.2 (53-2.1) and IL-22 (1H8PWSR) were from eBioscience. Isotype-matched antibodies (eBioscience) were used for control staining. All antibodies are used in 1:200 dilution in 1 × 10⁶ cells per 100 μl except Gr-1, Ly6C, I-Ab and CD45 (used in 1:500 dilution).

Flow cytometry was performed using the LSR Fortessa or FACS Canto II (BD Bioscience) and analyzed using FlowJo software (Tree Star, Ashland, OR). Dead cells were excluded by 7-AAD staining. Non-specific antibody binding was blocked with anti CD16/32 antibody. Fluorescence-conjugated mAb against CD11b (M1/70), CD11c (N418), F4/80 (BM8), Ly6C (HK1.4), MHC class II (I-A/I-E) (M5/114.15.2), CD103 (2E7), CD45 (30-F11), CD3 (17A2), CD4 (GK1.5), CD8α (53.67) were from eBioscience (Supplementary Table 5). Isotype-matched antibodies (eBioscience) were used for control staining. All antibodies were used at 1:200 dilution except CD11b (used in 1:100 dilution) and Ly6C, MHC class II, CD45 (used in 1:1000 dilution). The concentration of cell suspension was adjusted to 1 × 10⁶ cells per 100 μl.

BM-MSCs of passages 3–5 or transfected BM-MSCs were harvested and incubated with anti-CD90-FITC (11-0909), anti-CD90-PE (12-0909), anti-CD105-PE (12-1057), anti-CD73-APC (17-0739), anti-CD45-FITC (11-9459), anti-CD45-PE (12-9459), anti-CD34-PE (12-0349), anti-CD19-APC (17-0199), and anti-CD11b-PE (12-0113, eBioscience) antibodies at 4°C for 30 minutes. Results were detected by using an FC 500 MCL Flow Cytometer (Beckman Coulter). Appropriate isotype-matched antibodies (eBioscience) were applied as controls.

Cultured BMCs were flashed out from mouse long bones (femurs and tibias), and treated with 0.82 % NH₄Cl in PBS for 15 minutes at room temperature. The BMCs (1×10⁵ in 100 μl) were then incubated with PerCP-conjugated anti-CD4 antibody (1 μg; eBioscience) and then R-PE-conjugated anti-IL-17 (eBioscience) and APC-conjugated anti-interferon (IFN) gamma (eBioscience) antibodies (1 μg each). Isotype-matched antibodies (eBioscience) were used as controls. The number of CD4⁺IL-17⁺IFNγ⁻ Th17 cells per 1×10⁴ cells was measured on the flow cytometer.