Transcription factor fixation permeabilization solution

Single-cell splenocyte suspensions were obtained as previously described (Becker-Herman et al., 2011 (link)) and incubated with fluorescence-labeled Abs for 20 min at 4°C. Intracellular cytokines were assessed by plating 10⁶ cells/well in a 96-well plate and stimulating them for 5 h at 37°C with 5 µg/ml PMA (EMD Millipore), 1 µg/ml ionomycin (EMD Millipore), and GolgiStop (1:1,500 dilution; BD), after which intracellular staining was performed. Data were collected on a flow cytometer (LSR II; BD) and analyzed using FlowJo software (Tree Star). For human studies, cells were stained with a fixable viability dye for 10 min at room temperature followed by incubation with human TruStain FcX (BioLegend) for 10 min at 4°C. Surface Abs were stained for 20 min at 4°C followed by incubation with a transcription factor fixation/permeabilization solution (BD) for 40 min at 4°C. Intracellular Abs and transcription factor Abs were then stained for 40 min at 4°C in a permeabilization buffer (BD). Data collected with human samples were run on a cell sorter (FACSCanto II; BD).

For mouse studies, single-cell splenocyte suspensions were obtained, as previously described (Becker-Herman et al., 2011 (link)) and incubated with fluorescence-labeled antibodies for 20 min at 4°C. Data were collected on a LSR II (BD) and analyzed using FlowJo software (Tree Star). For human studies, cells were stained with a fixable viability dye for 10 min at room temperature, followed by incubation with human TruStain FcX (BioLegend) for 10 min at 4°C. Surface antibodies were stained for 20 min at 4°C followed by incubation with transcription factor fixation/permeabilization solution (BD) for 40 min at 4°C. Intracellular antibodies were then stained for 40 min at 4°C in permeabilization buffer (BD). Data collected with human samples were run on a FACSCanto II (BD).