For immunophenotypic analysis, the Flk-1+ MSCs were detached and washed with phosphate-buffered saline (PBS) containing 0·5% BSA (Sigma) and incubated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-labelled primary antibodies for 30 min at 4°C. To detect intracellular antigens, we fixed cells in 4% paraformaldehyde for 15 min at 4°C and then permeabilized them with 0·1% saponin (Sigma) for 1 h at room
temperature. Same-species, same-isotype FITC-labelled or PE-labelled immunoglobulin (Ig)G1 were used as negative controls. Finally, cells were washed twice with PBS containing 0·5% BSA and resuspended in 0·5 ml PBS for fluorescence activated cell sorter (FACS) analysis. Analysis was performed on a Beckman Roxadustat purchase Coulter flow cytometer. DBA-1 mice were obtained from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences. Collagen-induced arthritis (CIA) was produced in 8–10-week-old male DBA-1 mice, as described previously . Briefly, bovine type
II collagen (CII; Sigma) was emulsified with an equal volume of Freund’s complete adjuvant. Mice were injected at the base of the tail with 100 µl of emulsion containing 100 µg of collagen. On day 21, the mice received a booster injection of collagen emulsion in Freund’s incomplete adjuvant. Flk-1+ MSCs (1–2 × 106 cells in 0·3 ml PBS) were infused intravenously on either day 0 (day 0 group) or day 21 (day 21 group). Development of CIA was assessed twice a week. Paw swelling selleck kinase inhibitor was assessed by measuring the thickness of the hind-paws using a caliper. For better comparison, we averaged the paw swelling increase on days 32, 35, 39, 43 and 49, respectively, which took into account most of the disease process. The symptom score Ergoloid was assessed using the following system, as reported previously ; briefly, grade 0: no swelling; grade 1: ≥ 0·1 mm increase in paw swelling; grade 2: ≥ 0·2 mm increase in paw swelling; grade 3: extensive swelling (≥ 0·3 mm
increase in paw swelling) with severe joint deformity; and grade 4: pronounced swelling (≥ 0·45 mm increase in paw swelling) with pronounced joint deformity. On day 50, mice were anaesthetized for radiographic examination. The mice were then killed and limbs were fixed in 10% formalin, sections were prepared, and stained with haematoxylin and eosin (H&E) for histological assessment. Splenocytes were isolated by mechanical dissociation, followed by red blood cell lysis [10 min in NH4Cl (8·4 g/l)/potassium hydrogen carbonate (KHCO3) (1 g/l) buffer at 4°C]. Responder splenocytes were cultured in RPMI-1640 medium containing 10% FBS, incubated with 5 µg/ml concanavalin A (ConA; Sigma) for T cell stimulation or 25 µg/ml lipopolysaccharide (LPS; Sigma) for B cell stimulation. MSCs (10 Gy irradiated) were added to the splenocytes at ratios of 1:10 or 1:100.