Facsaria 2 flow cytometer

This assay was performed according to the method reported by Archin et al. [25 (link)]. Briefly, cells were fixed and permeabilized with Phosflow Fix Buffer I and Phosflow Perm Buffer II (BD Biosciences) according to the manufacturer’s protocol. The cells were then washed in staining buffer (PBS with 2% FBS and 0.1% sodium azide), blocked with 8% normal goat serum (Invitrogen), and incubated with anti-AcH3 (1:200 dilution; catalog no. 06–599, Millipore) or control rabbit IgG in blocking solution for 60 minutes at room temperature. Next, the cells were washed twice with staining buffer and incubated with goat-anti-rabbit IgG FITC- or Cy3-conjugated secondary antibody (1:200 dilution; Millipore) in staining buffer for 30 minutes at room temperature in the dark. Following a final wash, the cells were analyzed by flow cytometry using a FACSAria II flow cytometer and FlowJo software (Tree Star).

Plates were coated with CEA and blocked as above before the addition of 3 × 10⁵ CAR-transduced T cells per well in the presence of brefeldin A (10 μg/ml; Sigma-Aldrich), GolgiStop (0·7 μl/ml; BD Biosciences) and anti-CD107a-FITC. After incubation for 6 h at 37°C, cells were washed in FACS buffer (PBS/1% FCS) and incubated for 10 min at room temperature with LIVE/DEAD® Fixable Aqua stain (Life Technologies). Cells were then surface-stained with anti-CD4-PECy5·5 and anti-CD8-QD705, washed twice in FACS buffer, permeabilized using a Cytofix/Cytoperm kit (BD Biosciences) and stained intracellularly with anti-FLAG-PE, anti-IFN-γ-V450, anti-IL-2-APC and anti-TNF-α-PECy7. Data were collected on a custom-modified FACSAriaII flow cytometer and analysed with FlowJo software version 9·5.3 (TreeStar Inc.). Graphics were generated using the spice software suite [16 (link)].

Cells were prepared as reported elsewhere incubated with antibodies specific for CD45 (30F11; eBioscience), EpCAM (G8.8; DSHB, University of Iowa), MHCII (M5/114.15.2; BioLegend), Ly51 (6C3; BioLegend), UEA‐1 (Reactolab), Pdpn (8.1.1; BioLegend), Aire (5H12; eBioscience), and BrdU. For intracellular staining, cells were fixed, permeabilized (Cytofix/Cytoperm Kit, BD Biosciences), and labeled for the expression of Aire or the incorporation of BrdU. Stained samples were acquired on a FACSAria II flow cytometer and the data were analyzed using the FlowJo (Treestar) software.