Anti cd4 fitc clone rpa t4

Cultured T cells (2 × 10⁵) were incubated for 4 h at 37°C in 6.5% CO₂ with HPV OPPs, peptide matrices, minimization peptides, or defined peptide epitopes in the presence of GolgiPlug (BD PharMingen). After incubation, the T cells were washed and labeled with anti–CD4-FITC (clone RPA-T4; BD Biosciences) and anti–CD8-PerCP-Cy5.5 (clone RPA-T8; eBioscience) antibodies and LIVE/DEAD Fixable Near-IR Dead Cell Stain (Life Technologies). Cytofix/Cytoperm solution (BD PharMingen) was used to fix and permeabilize the T cells before washing and incubation with anti–IFN-γ–PE antibody (clone B27; BD Biosciences). The T cells were washed and resuspended in phosphate-buffered saline containing 1% paraformaldehyde and acquired using a BD LSRFortessa flow cytometer (BD Biosciences). For each sample screening or minimization or all other ICS assays, an internal positive control in which T cells are stimulated with a polyclonal stimulator (PMA/ionomycin) and also appropriate negative controls (no peptide) were included. Postacquisition analysis was conducted using FlowJo software (FlowJo LLC). Antigen-specific IFN-γ⁺ CD4⁺ and CD8⁺ responses were calculated by subtracting the proportion of IFN-γ⁺ cells in the no-peptide control from each peptide-stimulated response; ≥1% IFN-γ⁺ was considered an antigen-specific response.

Peripheral blood mononuclear cells (1 × 10⁵) were suspended in PBS supplemented with 20% human AB serum and then incubated for 30 minutes on ice with the appropriate dilution of the Ab anti‐CD4‐FITC (clone RPA‐T4), anti‐CD8‐PerCP‐Cy5.5 (clone RPA‐T8), or anti‐CD279 (PD‐1)‐APC (clone MIH4); all of these Abs including isotype‐matched negative controls were purchased from BD Biosciences. The stained cells were analyzed on a BD FACSCanto II flow cytometry system with FACS Diva software (BD Biosciences).