Pe cy7 anti human ifn γ

After treated with surface antibodies at 4°C for 30 minutes, cells were prepared with Transcription Factor Buffer Set (BD Biosciences) according to the manufacturer’s instructions for intracellular staining, and then incubated with intra-nuclear antibodies for 45 min. Cells were collected with a Cytek Athena (Cytek Biosciences). FlowJo software (Tree Star) were used to analyze the flow cytometry results. The blank control (cells unstained), single-stained control (PC5.5, APC-CY7, PE-cy7-A, APC-A, PE and FITC), and Fluorescence-Minus-One (FMO) control were set. The panel was designed as PC5.5 (CD8), APC-CY7 (CD4), APC-A (LEPR second antibody), PE (perforin), FITC (Granzyme B), and PE-cy7 (IFN-γ). The following antibodies were used: Alexa Flour 647 Donkey anti-rabbit IgG (BioLegend), Rabbit Anti-Leptin receptor antibody (bs-0410R, Bioss), APC-Cy7 anti-human CD4 (BD Biosciences), Percp-Cy5.5 anti-human CD8 (BD Biosciences), PE anti-human Perforin (BD Biosciences), FITC anti-human/mouse Granzyme B (Granzyme B, BioLegend), PE-Cy7 anti-human IFN-γ (BioLegend), Zombie Aqua Fixable Viability Kit (BioLegend), APC-Cy7 anti-mouse CD4 (Abcam), Percp-Cy5.5 anti-mouse CD8 (Abcam), PE anti-mouse Perforin (Abcam), FITC anti-mouse/human granzyme B (BioLegend), PE-Cy7 anti-mouse IFN-γ (Abcam).

Cells were first stained with LIVE/DEAD™ Fixable Aqua (Thermo Fisher, cat. L34957), followed by staining with antibodies against various surface markers. Samples were then acquired by a FACS instrument (CytoFLEX LX, Beckman Coulter). For intracellular staining, GolgiPlug (BD) was added upon at the beginning of T cell and target cell co-culture and cells were cultured for 6 h. Samples were then processed with the Cytofix/cytoperm kit (BD, cat. 555028) following the manufacture's protocol. The following antibodies were used: APC-eFluor 780 anti-human CD3 (Thermo Fisher, cat. 47003642), FITC anti-human CD8a (Biolegend, cat. 301006), BV605 anti-mouse TCRβ (Biolegend, cat. 109241), APC anti-human CD137 (Biolegend, cat. 309810), PECy7 anti-human IFN-γ (Biolegend, cat. 502528), Alx700 anti-human TNF-α (Biolegend, cat. 502928), PE anti-human IL2 (Biolegend, cat. 500307), PerCPCy5.5 anti-human CD107a (Biolegend, cat. 328616). HLA-A2/AFP158 tetramer was kindly provided by NIH Tetramer core facility at Emory University. All data were analyzed with FlowJo V10 software (FlowJo).