Cebinelli et al, Imaging-Enabled Spectral Flow Cytometry Panel for Profiling PD-1⁺/PD-L1⁺ Immune Cells interactions in humans (dataset_6_of_6)

This dataset supports the manuscript entitled "Imaging-Enabled Spectral Flow Cytometry Panel for Profiling PD-1⁺/PD-L1⁺ Immune Cells interactions in humans". This dataset contains imaging-enabled spectral flow cytometry raw data generated from peripheral blood leukocytes obtained from healthy adult human volunteers following informed consent and local ethical approval. Whole blood was collected in EDTA tubes and processed for leukocyte isolation by red blood cell lysis. Briefly, 500 µL of whole blood was transferred to 15 mL conical tubes, incubated with 10 mL of ammonium chloride-based lysis buffer for 5 min at room temperature, and centrifuged at 450 × g for 5 min. After supernatant removal, cells were washed once with DMEM, centrifuged again, and resuspended for counting. Leukocytes were adjusted to 1 × 10^5 cells per condition.

The dataset includes two main experimental groups: non-stimulated leukocytes and stimulated leukocytes. For the stimulated group, cells were cultured for 16 h at 37°C in a humidified incubator with 5% CO2 in DMEM supplemented with heat-inactivated fetal bovine serum, anti-CD3 (1 µg/mL), anti-CD28 (1 µg/mL), and IFN-γ (10 ng/mL). The non-stimulated group was cultured under the same conditions in medium alone and served as the control. These two biological conditions were used to compare basal and induced expression of PD-1 and PD-L1 across human leukocyte populations.

After culture, cells were stained for imaging-enabled spectral flow cytometry. Samples were first incubated with Fc block and Fixable Viability Stain 780, followed by staining with the optimised antibody panel. The final panel included CD5 BUV395, CD4 BV510, CD8 BV421, CD14 V450, CD15 APC, CD19 BV786, CD45 Pacific Orange, CD56 APC-R700, PD-1 PE-Cy7, and PD-L1 BB515. After staining, samples were washed, resuspended in staining buffer, and acquired on a BD FACSDiscover™ A8 instrument equipped with BD CellView™ technology.

In addition to the main biological groups, the dataset also includes control samples used for panel optimisation, spectral unmixing, and validation. These controls comprise unstained controls, single-stained controls prepared from stimulated and non-stimulated leukocytes, Fluorescence Minus Imaging (FMI) controls containing all non-imaging fluorochromes except those assigned to imaging channels, and Fluorescence Minus One (FMO) controls lacking either PD-1 or PD-L1. These controls were used to assess background signal, unmixing quality, spillover into imaging channels, and marker resolution.

Overall, this dataset provides raw and control data for the development and validation of an imaging-enabled spectral flow cytometry panel designed to identify major leukocyte subsets and to evaluate PD-1/PD-L1 expression and immune cell interactions in human peripheral blood.