Facscan flow cytometry system

Immunofluorescence analysis was performed as previously described [20] (link). Briefly, cells from passage 4 were fixed with 4% paraformaldehyde (CP, Shanghai, China) for 90 min, then, cells were washed with PBS for 5 min, incubated with 0.5% Triton X-100 (Sigma) at 37°C for 20 min, washed twice with PBS for 5 min each, blocked with 1% BSA at 37°C for 30 min, incubated with anti- rabbit antibodies: CD34-PE and CD44-PE (Bioss, Beijing, China), overnight at 4°C, and finally washed five times with PBS for 2 min each. Antibodies were replaced with PBS for negative control staining. CD34-PE and CD44-PE antibody distribution was observed with fluorescent microscopy (BX41TF, Olympus).
Flow cytometry was used to confirm surface antigen marker CD34 and CD44 expression. First, 1×10⁶ BMSCs were incubated with anti-CD34-PE, anti-CD44-PE for 30 min at 4°C in the dark. Labeled cells were washed, collected, and analyzed using the FACScan flow cytometry system (BD, Franklin Lakes, USA). Antibody was replaced with PBS for negative control staining.

cell‐cycle analysis was conducted according to a previously described procedure (Duan, Zhang, & Tong, 2001). Briefly, WI38 or 2BS cells, grown to approximately 85% confluency at PD45, were split in the ratio of 1:2. One of them was resuspended in DMEM containing different concentrations of BBR (0, 0.3125, 0.625, 1.25, and 2.5 μg/ml) and incubated at 37°C in 5% CO₂ for 72 hr, while the other one was resuspended in normal DMEM and incubated under similar conditions. The DNA content of the cells was measured by fluorescence‐activated cell sorting method using a Becton‐Dickinson FACScan Flow Cytometry System (BD). The data were analyzed using CellFIT software.

To confirm surface marker of MSCs, flow cytometry analysis was applied according to the manufacturer’s instructions. 1 × 10⁶ cells at P3 in the logarithm growth period were collected. After washing with 1% pre-cooled FBS/PBS and centrifuging at 350×g for 5 min, these cells were incubated with anti-CD45-APC (Invitrogen, USA), anti-CD29-FITC (Invitrogen, USA), and anti-CD44-APC (Novus Biologicals, USA) in the dark at 4 °C for 30 min, respectively. Labeled cells were washed twice and examined using the FACScan flow cytometry system (BD, Franklin Lakes, USA). FlowJo software (TreeStar, Ashland, OR, USA) was used to analyze the data. PBS solution was used as negative control. For cell cycle analysis of DNA content, the cells were cultured for 48 h under normoxia or hypoxia condition before they were collected, washed with PBS, and resuspended with 0.3 ml PBS and 1.2 ml pre-cooled 100 % ethanol for 1 h at − 20 °C. The cells were then centrifuged (300×g, 5 min) and resuspended with 1 ml PBS for 15 min at room temperature. The cells were then centrifuged (300×g, 5 min) again. One hundred microliters RNase A (Elabscience Biotechnology Co., Ltd., China) was added to each sample which was incubated at 37 °C for 30 min. Before test, 400 μl propidium iodide (Elabscience Biotechnology Co., Ltd., China) was added to each tube at 4 °C for 30 min.

First, THP1 cells were treated as mentioned above (cell lines and cell culture), then digested with accutase, collected, washed for three times with PBS. Then incubated with antibody mixture for 30 min (Brilliant Violet 421™ anti-CD163, BioLegend, 333611; FITC anti-CD86, BioLegend, 374203), washed with PBS again. A Bioscience FACScan Flow Cytometry System (BD Biosciences, Franklin Lake, NJ, USA) was used to detect markers expression.

Cell cycle analysis was conducted using the Beyotime Biotechnology Cell Cycle Kit and FACScan flow cytometry system (BD Company, New York, NJ, USA). HT-29 and NCM460 cells (1 × 10⁵ cells/well) in 6-well plates were treated with different concentrations (0, 100, 200, and 400 μg/mL) of SCS for 24 h. Proportions of cells in G2/M, S, and G0/G1 phase were quantified. Manufacturer protocols were followed for staining and analysis.

1 × 10⁶ cells were harvested after transfection for 24 h and resuspended in Annexin V binding buffer. 5 μl FITC-Annexin V and 5 μl PI were added to stain using the Apoptosis Detection Kit (BD Biosciences, San Jose, CA). Then, 400 μl PBS was added to the cells, which were analyzed using a FACScan flow cytometry system (BD Biosciences, San Jose, CA). Cell apoptosis was analyzed using FlowJo V7 software (Tree Star, Ashland, OR).

1 × 10⁵ transfected cells were seeded into each well of a 6-well plate and cultured in FBS-free medium for 48 h. Then, the cells were digested and resuspended to 1 × 10⁶ cells/ml with binding buffer. 5 μl FITC-Annexin V and 5 μl PI were added. Cell apoptosis was analyzed using FACScan flow cytometry system (BD Biosciences, San Jose, CA) and FlowJo V7 software (Tree Star, Ashland, OR).