Flow cytometry analyzer

Flow cytometry was performed as the previous report.¹⁸ Briefly, the third passage hucMSCs were harvested using 0.25% trypsin and washed twice using PBS. Then, hucMSCs were fixed using 4% paraformaldehyde (Sigma) and incubated with direct fluorescent label antibodies or isotype control antibodies as the Table S1 at 4°C for 30 min. The samples were detected by flow cytometry analyzer (BD, USA).

HUVECs were cultured in a hypoxia incubator and synchronized in the G0/G1 phase of the cell cycle, which was treated for 24 hours with gradient concentrations of FA (0.1 μg/ml, 1 μg/ml, and 5 μg/ml). At end of the period, cells were digested and washed three times with cold PBS (Beyotime technology, Shanghai, China), and then, the cell concentration was adjusted (1 × 10⁵/ml). Then, 70% ethanol was applied to fix cells overnight. In the end, the cells were stained with PI (propidium iodide) (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at 4°C (notice to avoid light). Fluorescence intensity was detected using a flow cytometry analyzer (BD Biosciences, San Jose, USA). The analysis of percentages of every phase (G1, S, and G2 phase) was accomplished with the GraphPad software.

To analyze cell surface maker expression, cells were incubated with specific antibodies for 30 min at room temperature. The flow cytometry analyzer (BD Biosciences) were used for acquiring the cells. The FACS data were analyzed with FlowJo v10 software.
Following antibodies were used: anti-CD4 (GK1.5, 100408, 1:250), anti-CD8a (53-6. 7, 100712, 1:250), anti-CD45 (30-F11, 03113, 1:250), Anti-CD3ε (145-2C11, 100326, 1:250), anti-CD11b (M1/70, 101206, 1:250), anti-F4/80 (BM8, 123116, 1:250), anti-Ly6G (1A8, 127654, 1:250), anti-Ly6C (HK1.4, 128008, 1:250) (all from Biolegend); anti-CD19(eBio1D3, 25-0193-82, 1:250), anti-NK1.1 (PK136, 11-5941-82, 1:250) (all from eBioscience).

The cell samples were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% TritonX-100 (Sigma). The samples were then labeled with primary or isotype control antibodies for 30 min at 4 °C. Primary and isotype control antibodies that were not conjugated to fluorophores were labeled with fluorophore-conjugated secondary antibody for 30 min at 4 °C. The labeled samples were detected by flow cytometry analyzer (BD, USA). Data analysis was performed on FCS Express 4 Flow Research Edition software.

For surface staining, cells were incubated with specific antibodies for 30 min at room temperature. The flow cytometry analyzer (BD Biosciences), FACSuite Software Bundle v1.0 (BD Biosciences), and FACSDiva Software v6.1 (BD Biosciences) were used for acquiring and analyzing the cells. The FACS data were analyzed with FlowJo v7.6.1 software. To perform intracellular cytokine staining, cells were stimulated with 100 ng/ml PMA and 500 ng/ml ionomycin, or 2 μg/ml GP_33-41 peptide together with GolgiPlug and GolgiStop (BD) for 5 h. Subsequently, cells were fixed and permeabilized, followed by staining with specific anti-cytokine antibodies.

To analyze cell surface maker expression, cells were incubated with specific antibodies for 30 min at room temperature. The flow cytometry analyzer (BD Biosciences) were used for acquiring the cells. The FACS data were analyzed with FlowJo v10 software.
Following antibodies were used: anti-CD4 (GK1.5, 100408, 1:250), anti-CD8a (53-6. 7, 100712, 1:250), anti-CD45 (30-F11, 03113, 1:250), Anti-CD3ε (145-2C11, 100326, 1:250), anti-CD11b (M1/70, 101206, 1:250), anti-F4/80 (BM8, 123116, 1:250), anti-Ly6G (1A8, 127654, 1:250), anti-Ly6C (HK1.4, 128008, 1:250) (all from Biolegend); anti-CD19(eBio1D3, 25-0193-82, 1:250), anti-NK1.1 (PK136, 11-5941-82, 1:250) (all from eBioscience).