Interferon γ pe

Analysis of samples was performed on a LSR2 and Fluorescence-activated Cell Sorting(FACS) on an ARIA or Fusion-instrument (BD Biosciences, San Jose, CA, USA). Single cells were gated on the basis of FSC-Height(FSC-H) and FSC-Area(FSC-A), and live cells were gated out using Propidium Iodide(PI) or the LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, MA, USA). Antibodies were obtained from BioLegend (San Diego, CA, USA): CD1a PB, CD3 Pe-Cy7, CD3 APC-Cy7, CD3 BV421, CD4 PerCp-Cy5.5, CD5 PE, CD8 Amcyan, CD8 APC-Cy7, CD14 PB, CD19-APC, CD27 APC-Cy7, CD33 PE, CD34 PerCp-Cy5.5, CD34 APC, CD45 Amcyan, CD45 Pe-Cy7, CD56 BV421, CD69 Pe-Cy7, CD137 PE, interferon-γ PE, IL-2 PB, TCRαβ-PE, and TCRγδ APC; BD Biosciences (San Jose, CA, USA): CD7-FITC; ThermoFisher Scientific (Waltham, MA, USA): CellTrace™ Violet Cell Proliferation Kit; and SCBT (Dallas, TX, USA): WASp-AF647.

Intracellular production of interferon γ and tumor necrosis factor α with CAR19 T cells or control non-transfected PBMCs following a 1-h co-culture with CD19-positive (Nalm6) and CD19-negative (TF-1) cell lines or control stimuli was performed as previously described.²⁸ (link)
Antibodies used for detection were CD3-BV711, CD4-BUV395, CD8-Pacific Blue, interferon-γ-PE, and tumor necrosis factor-α-PE/Cy7 (all from BD Biosciences).