Pacific blue conjugated anti cd3

Frozen PBMCs were thawed, washed and stained with the following combination of monoclonal antibodies, Pacific Blue-conjugated anti-CD3 (Biolegend), PE-Cy7-conjugated anti-CD56 (BD pharmingen), APC-Cy7-conjugated anti-CD16, PE-conjugated anti-NKG2A (Beckman Coulter), APC-conjugated anti-CD158a/h (Biolegend), APC-conjugated anti-CD158b1/b2/j (Biolegend), APC-conjugated anti-KIR3DL1 (Biolegend), Alexa Fluor 488-conjugated anti-NKG2C (R&D Systems), APC-Cy7-conjugated anti-CD27 (BD pharmingen), and PerCP-Cy5.5 conjugated anti-CD57 (Biolegend) monoclonal antibody. LIVE/DEAD Fixable Aqua was used for dead cell exclusion in every phenotypic analysis. The stained cells were then washed, fixed and permeabilized by BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) according to the manufacturer's protocol. Subsequently, cells were washed and stained intracellularly with FITC-conjugated anti-Ki67 (BD Pharmingen) for 30 min at 4°C. Cells were washed and re-suspended in PBS prior to flow cytometric analysis. Cells were acquired on FACS LSR-II and LSR Fortessa (BD Biosciences) and FACS data was analyzed by FlowJo v10 (BD). Samples with <20% live lymphocytes were excluded from the analysis.

FITC, PE, PE-cy5, APC, APC-cy7, or Pacific Blue conjugated anti-CD3, -CD4, -CD8, -CD44, and -CD62L, were purchased from BioLegend, eBioscience, and BD Pharmingen. Anti-LC3 (PD015) and -p62 were purchased from MBL. Anti-PI(3)P, -PI(3,4)P₂, -PI(3,4,5)P₃, and PI(3,4)P₂ lipid were purchased from Echelon Biosciences. IL-7, IL-4, and IL-15 were purchased from PeproTech. Acridine orange was purchased from Sigma. CytoID was purchased from Enzo Life Sciences. PIK75 was purchased from Cayman Chemical. iSHIP (AS1949490) and Dynasore were purchased from Tocris.

Mock-immunized mice and mice immunized with Pyfabb/f⁻ sporozoites as described above were injected intraperitoneally with 0.5 mg of anti-CD8 monoclonal antibody 2.43 (TIB210; American Type Culture Collection), anti-CD4 monoclonal antibody GK1.5, or an equivalent dose of rat IgG2b isotype control for two consecutive days before being challenged with plasmid DNA by HTVI. The dose and regimen was optimized to deplete >95% of CD8⁺ T cells or CD4⁺ T cells (data not shown). Depletion of specific cell types was confirmed by surface staining of PBMC with Pacific Blue-conjugated anti-CD3, PerCP/cy5.5-conjugated anti-CD8 and APC-conjugated anti-CD4 antibodies (Biolegend) by flow cytometric analysis one day before challenge.