Flow cytometric analyses were performed using an LSR II and/or LSR Fortessa X20. Data were analyzed using FlowJo (Tree Star). DAPI (0.5 mg/mL, Sigma-Aldrich) or a Live/Dead fixable cell stain (Ghost 780 Tonbo Biosciences) was used to exclude dead cells in all experiments, and anti-CD16/CD32 antibody (2.4G2) was used to block non-specific binding of antibodies via Fc-receptors. Flow cytometry antibodies are listed in Table S7 . Quantification of total cell numbers by flow cytometry was done using fluorescent beads (Biolegend Precision beads). For intracellular staining of IFNγ and IL-2 in vitro, cells were treated with Golgi Plug (Brefeldin A 500×) and were collected 4h later. Intracellular staining was performed in permeabilization buffer (eBioscience) for 30min and cells were subsequently analyzed by flow cytometry. Sorting of tumor cells after retroviral transduction was done using a BD FACSAria or a BD FACSAria Fusion. Purity of cell populations was determined by re-analysis of a fraction of sorted cell samples.
Precision beads
Manufactured by BioLegend
Precision beads are uniform, calibrated microspheres used for instrument calibration and performance verification. They provide a standardized means to assess the accuracy, sensitivity, and resolution of flow cytometers and other particle-based analytical instruments.
Automatically generated - may contain errors
2 protocols using precision beads
1
Flow Cytometric Analysis of Immune Cells
2
Flow Cytometric Analysis of Immune Cells
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Flow cytometric analyses were performed using an LSR II and/or LSR Fortessa X20. Data were analyzed using FlowJo (Tree Star). DAPI (0.5 mg/mL, Sigma-Aldrich) or a Live/Dead fixable cell stain (Ghost 780 Tonbo Biosciences) was used to exclude dead cells in all experiments, and anti-CD16/CD32 antibody (2.4G2) was used to block non-specific binding of antibodies via Fc-receptors. Flow cytometry antibodies are listed in Table S7 . Quantification of total cell numbers by flow cytometry was done using fluorescent beads (Biolegend Precision beads). For intracellular staining of IFNγ and IL-2 in vitro, cells were treated with Golgi Plug (Brefeldin A 500×) and were collected 4h later. Intracellular staining was performed in permeabilization buffer (eBioscience) for 30min and cells were subsequently analyzed by flow cytometry. Sorting of tumor cells after retroviral transduction was done using a BD FACSAria or a BD FACSAria Fusion. Purity of cell populations was determined by re-analysis of a fraction of sorted cell samples.
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