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).
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).
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