BV421 Mouse Anti-Human CD27

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	cells were transferred, centrifugation at 1500 g for 10 min and incubated with antibodies for 20 min at 37 °C. Annexin V was then added and stained for 15 min at room temperature.	After incubation, BMMCs were washed, centrifuged, measured by a FACSCanto II (BD Biosciences) and resulting data were analysed using FlowJo software.

Protocol tips
cells were transferred, centrifugation at 1500 g for 10 min and incubated with antibodies for 20 min at 37 °C. Annexin V was then added and stained for 15 min at room temperature.
Downstream tips
After incubation, BMMCs were washed, centrifuged, measured by a FACSCanto II (BD Biosciences) and resulting data were analysed using FlowJo software.

Publication protocol

Using the EasySep magnet to exclude infiltrating cells, adipocytes were stained with antibodies against anti-CD31 to identify endothelial cells, anti-CD105 for mesenchymal cells, anti-CD24 for stroma cells, LipidTox for neutral lipid droplets, and ant-CD36 for the differentiation stage of adipocytes (Fig. S3a). To analyse the cell survival of isolated BMMCs, cells were transferred, centrifugation at 1500 g for 10 min and incubated with antibodies against anti-human CD27 (BD Biosciences Cat#562513, RRID:AB_11153497), CD38 (BD Biosciences Cat# 563251, RRID:AB_2738097), Annexin V (BD Biosciences Cat# 556419, RRID:AB_2665412), anti-human CD3 (BioLegend Cat# 300407, RRID:AB_314061), anti-CD14 (BioLegend Cat# 325618, RRID:AB_830691), anti-human CD138 (Miltenyi Biotec Cat# 130-117-544, RRID:AB_2751398), CD19 (BD Biosciences Cat# 557791, RRID:AB_396873), and 7AAD (Milteny Biotec) for 20 min at 37 °C. Annexin V was then added and stained for 15 min at room temperature. After incubation, BMMCs were washed, centrifuged, measured by a FACSCanto II (BD Biosciences) and resulting data were analysed using FlowJo software.

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