Flow cytometry system

Apoptosis was detected using flow cytometry (FCM) with an Annexin V-FITC/PI double-staining kit. Briefly, cells were plated and treated for use in the cell viability assay. Following incubation for 24 h, the supernatant and attached cells were collected and stained with 10 μl of Annexin V-FITC and 5 μl of PI at 4°C for 20 minutes in the dark and analyzed using a flow cytometry system (Beckman Coulter, Fullerton, CA, USA).

For apoptosis detection, siCtrl, siZNF468#1 and siZNF468#2 MDA-MB-231 cells were harvested by EDTA-free trypsin. After staining with 3 μL PI and 5 μL Annexin V-APC (P-CA-207, Procell), the apoptosis was detected on the flow cytometry system (Beckman).

Nucleus pulposus cells were collected and detected using an Annexin V‐FITC Apoptosis Detection Kit (YEASEN) according to the manufacturer's instructions. Briefly, after washing, NP cells were resuspended and stained with Annexin V‐FITC and PI. Then, apoptotic cells were analysed using a flow cytometry system (Beckman Coulter).

For cell apoptosis, cells were transfected with siRNAs or plasmids were cultured for 48 h and harvested through trypsinization. Then, the cells were resuspended with PBS and the concentration of cells adjusted to 1 × 10⁶ cells/ml before staining. After staining the cells with propidium iodide and Annexin V-fluorescein isothiocyanate (Yeasen, Shanghai, China) for an hour on ice light aversion, cell apoptosis could be examined with a flow cytometry system (Beckman, Brea, CA, USA).
For cell-cycle detection, K1 and TPC1 cells transfected with siFOXP4-AS1 for 48 h were synchronized by starving in the G0/G1 phase, then fixed in precooled 75% ethanol and stained with propidium iodide (Yeasen). The DNA content of G0/G1, S, and G2/M phases in the cell cycle was examined through flow cytometry (Beckman). Triplicate independent tests were repeated.

To prepare the cells for flow cytometry sorting, live cells were harvested, resuspended in phosphate-buffered saline (PBS) with 10% fetal bovine serum and filtered using a 40 μm cell strainer (BD Falcon, Franklin Lakes, NJ, USA). Cell sorting was performed using a Beckman MoFlo Cell Sorting System (Beckman Coulter, Brea, CA, USA), and flow cytometry analysis was performed using a Beckman Cytomics FC500 Flow Cytometry System or a Gallios Flow Cytometry System (Brea, CA, USA).

After treatment, the cells were removed from the culture plates and washed in a centrifuge tube. First, 100 μL of CD16/32 (1:1000 dilution) was added to block the non-specific binding antibody. Then, 100 μL of APC-coupled anti-CD80 antibody (1:1000 dilution) and Brilliant Violet 421-coupled anti-CD206 antibody (1:1000 dilution) were double-stained at 37 °C in the dark for 20 min. After two steps of PBS washing, APC and Brilliant Violet 421 fluorophores were detected by a flow cytometry system (Beckman Coulter, Brea, CA, USA). For each sample, 3000 events were recorded.

Following treatment, the cells were collected, and apoptosis was detected by flow cytometry using an AnnexinV-FITC Apoptosis Detection Kit (Beyotime Biotech), following the manufacturer's instructions. Data acquisition and analysis were carried out using a flow cytometry system and related software (Beckman).