Apc conjugated anti cd73

CD73-positive cells were isolated from the subcutaneous fat tissue of 8-week-old C57BL/6-Tg CAG-EGFP mice. Similar to the isolation from human tissues, for collagenase treatments, the subcutaneous fat was chopped and minced in DMEM containing 2% FBS and 2 mg/mL collagenase (FUJIFILM Wako Pure Chemical) for 1 h at 37 °C with shaking. The cells were then diluted in HBSS containing 2% FBS, 10 mM HEPES, and 1% penicillin/streptomycin (Gibco). PI fluorescence was used to remove dead cells, and APC-conjugated anti-CD73 (Biolegend) was used for single-colour staining, followed by sorting with flow cytometry analysis on the FACS Aria II system (BD). The cells were cultured in MSCs medium as previously described^{14 (link)}. After culture for 14 days, non-adherent cells were removed, and the cells (7.0 × 10⁴ in 25 μL of PBS) were administered to the mice through transnasal injection 14 and 21 days after the first BLM administration. Control animals received an equal volume of PBS.

Human adipose tissue was treated with collagenase to dissociate it into single cells, and it was stained with APC-conjugated anti-CD73 (BioLegend, San Diego, CA, USA). To distinguish between living and dead cells, cells were suspended in propidium iodide solution, and this was followed by sorting using a FACSAria II (BD Biosciences). All experiments were analyzed using FlowJo software ver.10.8.1 (BD Biosciences). CD73⁺ cells were isolated from human adipose tissues as previously described (Suto et al., 2017 (link), 2020 ).
Human adipose-derived MSCs (CD73⁺ cells) were cultured in DMEM-Gluta MAX (Gibco) containing 20% FBS, 1% penicillin/streptomycin, and 20 ng/mL bFGF (REPROCELL, Kanagawa, Japan) as the MSC medium. Cells were grown to 70%–80% confluency in the MSC medium; following this, fresh medium containing LPS (500 ng/mL) or poly (I:C) (1 μg/mL) was added, and the cells were incubated for 1 h. Cells were washed twice in MSC medium. After 24 h, the conditioned media was collected. Cytokine levels were measured using the Cytometric Beads Array (BD Biosciences).