Cytoflex s flow cytometer

On day nine of NK cell culture, the purity of the NK cells was measure by flow cytometry. FACS buffer was prepared by adding fetal bovine serum (2%) into DPBS buffer. Samples of the culture were plated into two wells in a 96-well V-bottom plate and spun down at 300G for 5 min. One well was then stained with FACS buffer containing antibodies for detection of CD56 (PE-Vio770; 1:50) for 10 min in the dark at 4 °C. The plates were washed twice with FACS buffer before 100 μl of FACS buffer was added and analyzed immediately with CytoFLEX S flow cytometer (Beckman Coulter).
Transfection efficiency was assessed the day (18–24 h) post experiment by flow cytometry. FACS buffer was prepared by adding fetal bovine serum (2%) into DPBS buffer. Sample and negative control (no device, ND) cells were plated into a 96-well V-bottom plate and spun down at 300G for 5 min. The plates were then washed twice with FACS buffer before 100 μl SYTOX AADvanced dead cell staining solution (1:1000 SYTOX AADvanced diluted in FACS Buffer) were added and processed through a CytoFLEX S flow cytometer (Beckman Coulter).

Lung adenocarcinoma H522 (ATCC# CRL-5810), prostate carcinoma PC-3M (ATCC# CRL-1435), epidermoid carcinoma A431 (ATCC# CRL-1555), and triple-negative breast carcinoma MDA-MB-231 (ATCC# HTB-26) cells were seeded in a 12-well plate at 6 × 10⁴ cells per well. The cells were cultured in a CO₂ (ESCO, Horsham, PA, USA) incubator for 16–20 h, after which 150 μL of concentrated lentivirus was added to 350 μL of cell culture medium. After 16 h of incubation, the medium was changed for fresh growth medium. The transduction efficiency was assessed after 48 h using a CytoFLEX S flow cytometer (Beckman Coulter Inc., Brea, CA, USA) to confirm generation of H522(Kat+), PC-3M(Kat+), A431(Kat+), and MDA-MB-231(Kat+) cell lines. After that, some of the modified cells were transduced with lentivirus encoding the CD19 antigen. The transduction efficiency was assessed after 48 h using a CytoFLEX S flow cytometer (Beckman Coulter Inc., USA) to confirm generation of H522(Kat+CD19+), PC-3M(Kat+CD19+), A431(Kat+CD19+), and MDA-MB-231(Kat+CD19+) cell lines. In addition, H522(CD19+), PC-3M(CD19+), A431(CD19+), and MDA-MD-231(CD19+) lines were obtained by lentiviral transduction for further use in real-time biosensor cell analysis with xCELLigence system.

On day 7 of the experiment, multicolor flow cytometry was used to analyse the different forms of cell death. For this purpose, tumor cells were harvested with trypsin and 1 × 10⁵ cells per 96-well were incubated with AnnexinV (AxV)/propidium iodide (PI) staining solution (1.0 µg/mL of PI and 0.5 µg/mL of FITC-labeled AxV). Subsequent analysis was performed using a CytoFLEX S flow cytometer. The gating strategy is shown in Appendix A Figure A1. Cells being positive for both AxV and PI were defined as necrotic ones, with those being positive for AxV and negative for PI being defined as early and late apoptotic ones.