Clone 2d7

Functional grade-blocking anti-VCAM-1 (clone 429, eBioscience, 16-1061), ICAM-1 (clone YN1, eBioscience, 160541), VLA4/α4 integrin (clone R1-2, eBioscience, 16-0492), high+low-affinity LFA-1/CD11a (clone M17/4, eBioscience, 16-0111) and low-affinity CD11a (clone 2D7, BD, 553118), Rat IgG2a (clone eBR2a, eBioscience, 16-4321) and Rat IgG2b (clone eB149/10H5, eBioscience, 16-4031) were used to treat endothelial layers or T cells at 4 °C for 30 min at 10 μg ml⁻¹. In some experiments, low-endotoxin, no-azide (LEAF) grade anti-IL-10 (clone JES5-2A5, Biolegend, 504903), LEAF grade anti-TGF-β1 (clone 19D8, Biolegend, 521703) or LEAF Grade Rat IgG1k (RTK2071, Biolegend, 400413) were incubated with T cells at 10 μg ml⁻¹ for 30 min at 4 °C before transmigration. T cells were washed 2 × before use except in the case of anti-TGF-β1, anti-IL-10 and control Rat IgG1k, while VCAM-1 and ICAM-1 blockades of endothelial cells were also maintained during assays.

The mBMSCs were isolated from either 8–12 weeks old (young mBMSCs) (n = 3) or 78–104 weeks old (aged mBMSCs) (n = 3) C57Bl/6 mice by flushing the bone marrow from femurs and tibias. The bone marrow cells were rinsed twice in PBS and the nucleated cells were counted by Methyl Violet staining. The nucleated cells were cultured in Alpha-MEM Glutamax (Gibco, Milan, Italy) supplemented with 10% FBS (Gibco, Milan, Italy), 2 mM of L-glutamine, 100U/mL penicillin, 100 μg/mL streptomycin and 1 ng/mL of fibroblast growth factor-2 (FGF-2) (standard medium). The cells were maintained in an incubator at 37 °C, 5% CO₂, changing the medium every 3 days. Only cells from P1 or P2 passages were used for further analysis and secretome isolation. Both young and aged mBMSCs cultured in standard medium were analyzed for the expression of the following markers, using monoclonal antibodies against CD44 (Clone IM7, BD Biosciences, Milano, Italy), H2Kb (Clone AF6-88.5, BD Biosciences), CD105 (Clone MJ7/18, Biolegend, San Diego, CA, USA), CD29 (Clone HMb1-1, eBioscience, Waltham, MA, USA), CD90.2 (Clone 30-H12, BD Biosciences), CD45 (Clone 30-F11, BD Biosciences), CD31 (Clone 390, eBioscience), CD11a (Clone 2D7, BD Biosciences) and CD34 (Clone 4H11, eBioscience).