Gallios 10 3 flow cytometer

JEG3 cells treated with CER+Q-VD-OPh were dissociated using 0.5 ml per well TrypLE express (Invitrogen), washed and processed in accordance with the FITC Annexin V Apoptosis Detection Kit 1 (BD Pharmingen, San Jose, CA, USA) at a concentration of 1 × 10⁶ cells/ml. In brief, Annexin V and/or propidium iodide were added to the cells and incubated alongside a binding buffer for 15 min at room temperature protected from light, then thoroughly washed. Data were acquired using the Beckman Coulter Gallios 10/3 flow cytometer and analyzed with Kaluza flow cytometry software (Beckman Coulter, Brea, CA, USA). Annexin V-positive events were measured using the FL-1 channel (525 emission maximum), whereas the FL-4 channel (670 nm emission maximum) was used for propidium iodide-positive events. Unstained cells, cells stained only with FITC Annexin V and cells stained only with propidium iodide were included as controls.

DNA content measurement for analyzing cell cycle parameters was performed according to Kaltschmidt and colleagues [34 (link)] by harvesting 1 x 10⁶ cells at 300 g for 5 min followed by fixation with 70% (v/v) ethanol. After centrifugation at 300 x g for 10 minutes, staining solution (PBS containing 1 mg/ml glucose (Carl Roth GmbH), 4´,6-diamidino-2-phenylindole (DAPI; 0.5 mg/ml; Sigma-Aldrich), and 100 Kunitz units RNaseA (Thermo Fisher Scientific) was applied for 60 min under exclusion of light.
For apoptosis measurement, 1 x 10⁶ cells were labeled with Annexin V-PE (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. For analysis of H2B-mCherry, 1 x 10⁶c-REL^+/+ and c-REL^-/- cells were harvested and directly applied for flow cytometric analysis without additional staining procedures.
DAPI or Annexin V-PE-labeled cells as well as unstained cells (H2B-mCherry) were analyzed using a Gallios^™ 10/3 flow cytometer (Beckman Coulter, Brea, CA, USA). Data analysis was performed using FlowJo Software (TreeStar, Olten, Switzerland), doublet discrimination for cell cycle analysis was assured by appropriate gating strategies.

Purified CD4⁺ T cells or induced Th17 cells were stimulated for six hours with PMA (500 ng/ml; Abcam, Cambridge, UK) and ionomycin (1.5 μM; Cell Signaling Technology^®, Danvers, USA). Brefeldin A (eBioscience, San Diego, USA) was added to cell culture two hours before flow cytometry staining. Dead cells were excluded by the LIVE/DEAD™ Fixable Dead Cell Stain Kit (Invitrogen, Thermo Fisher Scientific, Carlsbad, USA). Briefly, cells were stained with cell surface targets including CD4, CD25, and CD127, then fixed and permeabilized by the BD Cytofix/Cytoperm Kit (BD Bioscience, Heidelberg, Germany) or the True-Nuclear™ Transcription Factor Buffer Set (BioLegend Inc., San Diego, USA) according to the manufacturer’s instructions and stained with anti-IL17A, anti-IFN-γ, and anti-FoxP3 (all antibodies and isotype antibodies from Bio Legend Inc., San Diego, USA). Th17 cells were identified by IL-17A expression in purified CD4⁺ T cells. Treg cells were defined as CD4⁺CD25⁺CD127⁻FoxP3⁺ T cells in humans and CD4⁺FoxP3⁺ T cells in mice. Flow cytometry was performed on the Gallios 10/3 flow cytometer and the results were analyzed by Kaluza Analysis Software (Both from Beckman Coulter, Krefeld, Germany).