Cytoflex system

Apoptosis of cells transfected with plasmids encoding flag-ORF7b or treated with inhibitors was detected using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Staining/Detection kit (BMS500FI-300; eBioscience, San Diego, CA, United States). Cells (1 × 10⁵) from each well were collected 18 h after transfection, and 500 μl of binding buffer was added to resuspend them after washing thrice with phosphate-buffered saline. Then, cells were mixed with 5 μl of Annexin V-FITC and 2 μl of PI (Propidium Iodide, PI). Then, the cells incubated in the dark for 10 min at room temperature. Cell apoptosis was detected by flow cytometry using a CytoFLEX^™ system (Beckman Coulter, Fullerton, CA, United States) and the experiment was repeated independently thrice.

The obtained neutrophils were collected through centrifugation at 300g for 10 min at 4 ℃, and the pellets were resuspended with 100 μL PBS containing 0.25 μg of the allophycocyanin (APC)-conjugated anti-mouse/human CD11b antibody (Biolegend, San Diego, CA, USA) and the phycoerythrin (PE)-conjugated anti-mouse Ly-6G antibody (Biolegend), followed by incubation on ice for 30 min. Levels of CD11b and Ly-6G in the neutrophils were analysed by flow cytometry using the CytoFLEX system (Beckman Coulter, Pasadena, CA, USA).

Cell cycle analysis was performed using the Cell Cycle Staining kit (70-CCS012, Multi Sciences, Hangzhou, China) according to the manufacturer’s protocol. For the cell apoptosis analysis, the Annexin V-FITC/PI Apoptosis Detection Kit (70-AP101–100, Multi Sciences) was used according to the manufacturer’s instructions. The stained cells were washed with PBS and analyzed via flow cytometry on a CytoFLEX System (CytoFLEX S, Beckman Coulter, Brea, California, USA).

mES cells were dissociated with trypsin/EDTA, resuspended in culture medium, centrifuged and resuspended in PBS. For intracellular flow cytometry, 0.5 ml of cold fixation buffer (BioLegend, 420801) was added and then incubated at room temperature for 10 min. Subsequently, the cells were labelled with the unconjugated rabbit DPY30 antibodies (Bethyl Laboratories, A304-296A) and subsequently with a FITC-conjugated goat anti-rabbit IgG antibody. Flow cytometry was performed using the Beckman Coulter CytoFlex system. The gating strategy is shown for the E14 sample in Supplementary Fig. 5.

Recombinant protein translocation to the bacterial surface via the MisL autotransporter was evaluated by immunofluorescence and flow cytometry, as previously described (34 (link), 35 (link)). Briefly, after inducing the expression of the recombinant proteins as described above, 10⁸ bacteria were incubated with antibody mouse anti-Flag FITC (1 mg/ml, Sigma-Aldrich) at a dilution of 1:100 in PBA 1× for 2 h, at room temperature, at 100 rpm, in dark conditions. The bacterial suspension was washed in PBS 1× and resuspended in 50 μl PBS 1×, 5 μl of which were used for fluorescent microscopy analysis (Olympus Microscope, model IX73), and the rest was resuspended in 450 μl PBS 1× for flow cytometry analysis in the CytoFLEX system (Beckman Coulter).

The following procedure was used for the flow cytometry analysis of the cell cycle stage distribution profile. Cell samples were taken from the culture, re-suspended in the isotonic solution (0.9% NaCl) and filtered (Filicons, by Becton Dickinson Biosciences, Franklin Lakes, NJ, USA, pore diameter 50 μ). Cell concentration in the resulting sample was normalized to 1 × 10⁶ mL⁻¹. Corresponding doses of the tested substance are given in μg per ml of the cell culture normalized for 1 × 10⁶ mL⁻¹.
Analysis was carried out using a CytoFLEX system (Beckman Coulter Inc., Brea, CA, USA), monitoring specific markers using cell cycle stage and apoptosis analysis assays by Beckman Coulter Inc., Brea, CA, USA. Prior to the analysis cells were re-suspended in the phosphate-buffered saline (PBS). During each test, 50,000 to 75,000 cells were analyzed. Results of the tests were analyzed and saved using the firmware of the flow cytometry machine.