Novocyte 3000ryb flow cytometer

Treated or control THP-1 or JY cells seeded into 24-well plates were labeled with Sytox Green Dead Cell Stain (Thermo Fisher Scientific) and AlexaFluor647-conjugated annexin V (Thermo Fisher Scientific) at dilutions of 1:1,000 and 1:20, respectively, in annexin binding buffer for 15 min at room temperature. Fluorescence intensities of individual cells were subsequently measured using a NovoCyte 3000RYB flow cytometer (ACEA Biosciences, San Diego, CA, United States). Sytox Green and AlexaFluor647 fluorophores were excited at 488 and 640 nm, respectively, and emitted intensities were measured using 530/30 and 660/20 emission filters, respectively. During data analysis, the fraction of Sytox Green and annexin V negative living cells was calculated for each sample using FCS Express.

Cell migration and invasion were assessed using an xCELLigence Real Time Cellular Analysis (RTCA) DP instrument (Acea Biosciences, San Diego, CA). Migration was measured using uncoated CIM-Plate 16 (Acea) plates. For invasion assessments, plates were pre-coated with a 1:20 dilution of Matrigel GFR (Corning). To monitor invasion and migration, cells were serum-starved for 16 h, collected by trypsinization, and plated at 200,000 viable cells per well in the top chamber of a CIM-Plate 16. Viable cell counting was performed using propidium iodide staining with quantitation on an Acea NovoCyte 3000 RYB flow cytometer. Ten percent of FBS-containing media was used as a chemoattractant in the bottom chamber. Cells were allowed to settle for 10 min at room temperature prior to loading CIM-Plates onto the xCELLigence DP. Impedance data were acquired at 15-min intervals for 40 h (migration) and 60 h (invasion). Proliferation was assessed using an xCELLigence RTCA MP instrument (Acea). Transduced cells were plated at 20,000 viable cells per well in E-Plate View 96 plates, allowed to settle for 10 min at room temperature, and loaded onto the xCELLigence MP, and impedance data acquired at 15-min intervals for 60 h.