Cytofix cytoperm plus fixation permeabilization kit with golgiplug protein transport inhibitor containing brefeldin a

Tumors were collected (n = 3–5/group) from control and HER3-DC1–treated TUBO tumor–bearing mice. Tumor-infiltrating lymphocytes (TIL) were isolated following the protocol described above and cocultured with mature DC1 cells pulsed with individual HER3 peptides (10:1 TIL:DC; i.e., 10⁶ TIL:10⁵ DC in 1 mL total volume). Intracellular staining was performed using the BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit with BD GolgiPlug Protein transport inhibitor containing Brefeldin A (cat. #555028, BD Biosciences). Briefly, 6 hours after TIL:DC coculture, GolgiPlug was added to inhibit intracellular protein transport (1 μL/10⁶ cells) for 12 hours. Cells were harvested the next day, and surface staining with CD45-BUV395 (cat. #564279, Clone 30-F11, BD Biosciences), CD4-BUV805 (cat. #612900, Clone GK1.5, BD Biosciences), and CD8-Pacific Blue (cat. #558106, Clone 53–6.7, BD Biosciences) was performed as described above. Cells were fixed and permeabilized following the manufacturer’s protocol and were stained for intracellular IFN-γ-PE (cat. #554412, Clone XMG1.2, BD Biosciences). Acquisition was performed using an LSRII (BD Biosciences) cytometer, and FACS data analysis was performed with FlowJo software (FlowJo).