NCT04013854 Neoadjuvant anti-PD-1 Nivolumab Clinical Trial (To Patient 67)
Description
Flow cytometry data for NCT04013854 Neoadjuvant anti-PD-1 Nivolumab Clinical Trial. Antigen-specific profiling identifies T-bet+ melanoma-specific CD8+ T cells associated with pathologic response to neoadjuvant anti-PD-1 therapy Cancer Cell 2025
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Combinatorial tetramer flow cytometry staining Single-cell suspensions prepared from blood and tissue samples were treated with DNase I, washed with FACS staining buffer (PBS supplemented with 2% fetal bovine serum), and aliquoted into U-bottom 96-well plates for staining. Both staining samples and non-staining controls were included. Tetramer Staining: Tetramers were diluted in FACS staining buffer containing Brilliant Stain Buffer (BD Biosciences; used to reduce spectral overlap) and milk to prevent tetramer aggregation. A total of 50 µL of diluted tetramer solution was added to the staining samples, while 50 µL of tetramer-free buffer was added to the non-staining controls. The plates were incubated for 20 minutes at room temperature in the dark. Following incubation, the samples were washed with 150 µL of FACS staining buffer, centrifuged at 2,000 rpm for 3 minutes, and the supernatant was discarded. Surface Staining: Surface antibodies and live/dead viability dyes were diluted in FACS staining buffer supplemented with Brilliant Stain Buffer. A total of 50 µL of diluted surface staining solution was added to the staining samples, while 50 µL of surface stain-free buffer was added to the non-staining controls. The samples were incubated for 20 minutes at room temperature in the dark, followed by a wash with 150 µL of FACS staining buffer, centrifugation at 2,000 rpm for 3 minutes, and removal of supernatants. Fixation and Permeabilization: Fixation and permeabilization were performed using commercially available buffer set (InvitrogenTM eBioscienceTM Foxp3 / Transcription Factor Staining Buffer Set) prepared according to the manufacturer's instructions. A total of 50 µL of fixation/permeabilization buffer was added to each sample, and the plates were incubated for 20 minutes at room temperature in the dark. The samples were then washed with 150 µL of permeabilization buffer, centrifuged at 2,000 rpm for 3 minutes, and supernatants were removed. Intracellular Staining: Intracellular antibodies were diluted in permeabilization staining buffer containing Brilliant Stain Buffer. A total of 50 µL of diluted intracellular staining solution was added to the staining samples, while 50 µL of intracellular stain-free buffer was added to the non-staining controls. The plates were incubated overnight at 4°C in the dark. Following incubation, the samples were washed with 150 µL of permeabilization buffer, centrifuged at 2,000 rpm for 3 minutes, and supernatants were removed. Finally, both staining samples and non-staining controls were resuspended in 200 µL of 1% paraformaldehyde solution for fixation before flow cytometric acquisition.
Institutions
- University of Pennsylvania