Apc conjugated anti cd39

Cells from the BALF were stained with surface markers, including PE-Cy7 conjugated anti-CD45 (BD Biosciences, CA, USA), AF488 conjugated anti-CD11b (BD Biosciences, CA, USA), and PE conjugated anti-Ly6G (BD Biosciences, CA, USA). Cells from blood and spleen were stained with various surface markers, including PE-Cy7 conjugated anti-CD45 (BD Biosciences, CA, USA), Percp-cy5.5 conjugated anti-CD3 (BD Biosciences, CA, USA), FITC conjugated anti-CD4 (BD Biosciences, CA, USA), PE conjugated anti-CD25 (BD Biosciences, CA, USA), and APC conjugated anti-CD39 (BD Biosciences, CA, USA).
Peripheral blood mononuclear cells (PBMCs) from ARDS patients and healthy donors were stained with PerCP conjugated anti-CD3 (BD Biosciences, CA, USA), FITC conjugated anti-CD4 (BD Biosciences, CA, USA), APC conjugated anti-Foxp3 (BD Biosciences, CA, USA), and PE conjugated anti-CD39 (BD Biosciences, CA, USA). This study was approved by the Jinling Hospital Ethics Review Committee and written informed consent was provided by all subjects or their legal representatives.

Tregs from mouse spleens and blood were isolated by a CD4⁺CD25⁺ Regulatory T-cell Isolation Kit (Miltenyi Biotec, GEM) according to the manufacturer’s recommendations. The Tregs were stained with APC conjugated anti-CD39 (BD Biosciences, CA, USA) and subjected to flow cytometry.
Tregs isolated from WT and CD39^−/− mice were stimulated in vitro for 3 days with 4 ng/ml recombinant mouse IL-2 (Absin, CHN) in 24-well plates, which were precoated with 1 μg/ml anti-mouse CD3 (Absin, CHN) and anti-mouse CD28 Abs (Absin, CHN). The purity of the isolated Tregs was >95% by a flow cytometric analysis with FITC conjugated anti-CD4 (BD Biosciences, CA, USA) and PE conjugated anti-CD25 (Miltenyi Biotec, GEM).