Apc cy7 mouse anti human cd4

Live-cell immunofluorescence measurements of cell surface expression for CD4 was performed on a Sony SH800 Cell Sorter. All staining and washes were done with cells suspended in phosphate-buffered saline (PBS) containing 2% hiFBS (heat-inactivated fetal bovine serum) and 0.1% sodium azide, chilled on ice. For each sample, one million cells were first resuspended in100 μL buffer containing 5 μL Fc Receptor Blocking Solution (Biolegend, #422302) and incubated for 15 minutes. Cells were then spun down and resuspended in 100 μL of buffer containing fluorescently conjugated antibodies for one hour (5 μL of each antibody per sample): APC-Cy7 Mouse Anti-Human CD4 (1:20 dilution; BD Biosciences, #557871), APC-Cy7 Mouse IgG1, κ Isotype Control (1:20 dilution; BD Biosciences, #557873). Following staining, the samples were washed three times by resuspending in 300 μL of fresh buffer, chilled on ice. Following collection of flow cytometry data,.fcs files were exported and processed using the Python package FlowCytometryTools^{111 (link)} (v. 0.5.1).

The functionality of peptide-specific T cells was analysed by ICS. PBMCs were stimulated with individual peptides at a final concentration of 10 µg ml⁻¹ and incubated for 12 h in the presence of GolgiStop (BD Biosciences), Brefeldin A (Sigma-Aldrich), and FITC mouse anti-human CD107a mAb (1:100, clone H4A3, BD Bioscience). Ionomycin and PMA (Sigma-Aldrich) served as a positive control. Thereafter, cells were stained for surface markers with PerCP anti-human CD8a (1:100 dilutions, clone RPA-T8, BioLegend) and APC-Cy™7 mouse anti-human CD4 (1:100, clone RPA-T4, BD Bioscience) mAb in addition to using LIVE/DEAD™ Fixable Aqua Stain to discriminate viable cells (1:400 dilutions, Invitrogen, Waltham, MA, USA). After incubation, a fixation/permeabilization step with Cytofix/Cytoperm solution (BD Biosciences) was added and the cells were further stained with PE anti-human IFN-γ (1:200 dilutions, clone B27, BioLegend) and Pacific Blue™ anti-human TNF-α (1:120, clone MAB11, BioLegend) mAb. Data were acquired by BD FACSCanto II flow cytometry (BD Biosciences) using FACS DIVA software (BD Biosciences). The gating strategy applied for the evaluation of flow cytometry-acquired data is provided in Supplementary materials (suppl. Figure 1).