Fortessa flow cytometry

After gathering the transfected HeLa and CaSki cells, the precooled PBS was adopted to rinse the cells. To measure cell apoptosis, Annexin V-FITC/PI Apoptosis kit obtained from BD Biosciences (San Jose, CA) was exploited. After culturing for 15 min in dark, cells were subjected to analysis with fortessa flow cytometry (BD Biosciences, CA. USA). Three experiments were conducted for each sample.

Lung tissues of mice were prepared into cell suspension, and appropriate amount of surface marker antibodies were added according to the instructions, and incubated for 30 min on ice, protected from light. Flow cytometry data were obtained using Fortessa flow cytometry (BD FACSCantoII) and analyzed using FlowJo software (Becton Dickinson). Single-cell suspensions were stained with combinations of the following antibodies (Biolegend): anti-CD3 (APC/Cyamine7), anti-CD4 (FITC), anti-F4/80 (APC), anti-CD49b (PE), anti-CD11c (PE), and anti-CD19 (Percp/Cyamine7).

Following transfection for 24 h, cells were collected by treating them with EDTA-free trypsin at 300 g and centrifuging them for 5 min at 4 °C. The cells were then washed twice with pre-chilled PBS, each time at 300 g and centrifuged for 5 min at 4 °C. Subsequently, 1–5 × 10⁵ cells were collected and resuspended using 100 μL of L1 × Binding Buffer. Annexin V-647 (5 μL) and PI (5 μL) were added per tube, which was then incubated for 15 min on ice before analyzing immediately using BD Fortessa flow cytometry. The data collected was analyzed using BD FACSDiva™ software (BD Biosciences) and Flowjo v10 software (Flowjo).

Cells were seeded in 100 mm dishes at a density of 0.5 × 10⁶ cells. After 72 h of treatment with triptolide, cells were trypsinized and centrifuged at 300× g for 5 min. The unattached cells were collected from the growth media and pooled. The cells were washed and re-suspended in PBS. The cells were fixed with absolute alcohol, replicates were accumulated, and stored at 4 °C in absolute alcohol until analysis [33 (link)]. On the day of analysis, the cells were centrifuged and re-suspended in propidium iodide (PI/RNase, Cat. # 4087S; Cell Signaling, Boston, MA, USA). Cells with the PI/RNase were incubated at 37 °C for 45 min. After incubation, cells were centrifuged, washed, and re-suspended in PBS. Data acquisition was carried out on BD Fortessa flow cytometry. Forward and side scatter area gating were used to identify singlets. Doublets were also discriminated by using forward scatter area and forward scatter height. Interval gates were placed on the detected peaks representing the phases of cell cycle on a PI histogram plot. Percentage of cells were estimated from each gate representing sub-G₀/G₁, G₀/G₁, S, G₂/M phases of cell cycle.

On day 10 of PMN mice model, lung tissues were harvested from different treatment groups and mechanically minced and digested to obtain the single-cell-suspensions as described above. The single-cell suspensions washed with PBS and resuspended were incubated with APC-antimouse-CD11b (1:250, Cat. 101211, Biolegend, USA) and PE/Cy7-antimouse-Ly6g (1:150, Cat. 127617,Biolegend, USA) antibodies in 100 μl 1% BSA for 30 min at 4 °C in dark. After centrifuged at 400 × g and washed with PBS, cell pellets were analyzed by BD Fortessa flow cytometry. The data were analyzed using FlowJo software v10.6.2.