In cell analyzer software

TY10 cells are adult human brain microvascular endothelial cells (BMECs) immortalized with temperature‐sensitive SV40 large T antigen (tsA58), as previously described.12 All of the analyses were performed 2 days after a temperature shift from 33 to 37ºC. The cells were cultured in medium containing IgG (final concentration, 500 µg/mL) obtained from patients with NMOSD or healthy controls after substitution for serum‐free MCDB 131 medium for 1 h for the NF‐κB p65 analyses; the primary Abs (NF‐κB p65 rabbit monoclonal antibody [mAb]) and secondary Abs (Alexa Fluor 488 goat anti‐rabbit IgG) were previously described.
TY10 cells were fixed with 4% paraformaldehyde (PFA), washed and then permeabilized with 0.3% Triton X‐100. After blocking overnight in 5% FBS/0.3% Triton X‐100 in PBS, the cells were incubated with primary Abs, followed by the secondary Abs at room temperature.
Five thousand cells per well were plated in Greiner CELLSTAR® 96‐well plates (Greiner), then immunostaining for NF‐κB p65 was performed. The plates were scanned, and images were captured by an In Cell Analyzer 2000 (GE healthcare) at × 20 magnification with six fields of view per well (equivalent to 800–1000 cell events). The images were then analyzed with the In Cell Analyzer software program (GE healthcare). The data represent the mean value of triplicate experiments.

CHO cells were cultured and plated at 3,000 cells in 100 μl media per well into black, clear-bottom 96-well plates (Greiner Bio-one). After overnight incubation at 37 °C and 5% CO₂, cells were treated accordingly with compounds, and subjected to immunostaining for catalase and GO. Briefly, cells were fixed in 4% paraformaldehyde for 20 min, rinsed with PBS, permeabilized with 0.3% Triton X-100 for 15 min, and followed by blocking with cell staining buffer (420201, BioLegend) for 60 min. Cells were then incubated with 1:50 dilution of primary antibodies against catalase (sc-50508, Santa Cruz Biotechnology) and against GO (sc-85589, Santa Cruz Biotechnology) at room temperature for 2 h. After washing with PBS, anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (A11034, Life Technologies) and anti-goat secondary antibody conjugated with Alexa Fluor 594 (A11058, Life Technologies) were added in 1:250 dilution for 1 h. Cells were then stained for the nucleus with Hoechst 33342 (H3570, Life Technologies) for 20 min and finally imaged for tri-color fluorescence using INCell Analyzer 2000 (GE Healthcare) with 40x objective lens, and through FITC, Texas Red and DAPI filter sets. Microscopy images were processed in INCell Analyzer software (GE Healthcare).