Pe conjugated anti human ifn γ clone 4s b3

FITC-conjugated anti-human Vδ2 TCR (clone B6), allophycocyanin-Cy7-conjugated anti-human CD3 (clone SK7), FITC-conjugated anti-human TCR γδ (clone B1), allophycocyanin-conjugated anti-human Vδ2 mAb (clone B6), FITC- and PE-conjugated anti-human CD3 (clone SP34), PerCP-Cy5.5 anti-human CD27 (clone MT271), PE-Cy7-conjugated anti-human CD28 (clone CD28.2), allophycocyanin-conjugated anti-human CD45RO (clone UCHL1), PE-conjugated anti-human IL-21 (clone 3A3-N2.1), PE-Cy5- and PE-conjugated anti-human CD107a (clone H4A3), PE-conjugated anti-human IFN-γ (clone 4S.B3), and allophycocyanin-conjugated anti-human TNF-α (clone MAB11) antibodies were purchased from BD Biosciences (San Jose, CA). PE-conjugated anti-human CD107a (clone H4A3), PerCP-Cy5.5-conjugated anti-human IL-17 (clone eBio64DEC17), PE-conjugated anti-human granzyme B (clone GB11), PE-conjugated anti-human IL-4 (clone 4D9-8), and PE-conjugated anti-human perforin (clone dG9) antibodies were purchased from eBioscience (San Diego, CA). The PE-conjugated anti-human IL-22 (clone 142928) antibody was purchased from R&D Systems (Minneapolis, MN). Live cells were distinguished from dead cells by staining with Hoechst 33258 or Live/Dead Blue (ThermoFisher Scientific, Waltham, MA). Pamidronate (3 mg/ml) was from Hospira, Inc. (Lake Forest, IL). Zoledronate was provided by Dr. Eric Oldfield.

Cells from DC-peptide-PBL cocultures were harvested and coincubated with the indicated peptide (20 μg/mL) or without peptide (negative control) for 16 h in serum-free RPMI 1640 medium in a 48-well plate at 37 °C and 5% CO₂. Next, a BFA/monensin mixture was added to the cells for another 6 h of culture. Cells were then harvested, washed, blocked with human FcR Blocking Reagent (MACS Biotech) for 20 min at 4 °C, and stained with FITC-labeled anti-CD3 and APC-labeled anti-CD8 antibodies for 30 min at 4 °C. After washing, the cells were fixed, permeabilized, and further incubated with PE-conjugated anti-human IFN-γ (clone 4S.B3, BD) for another 30 min at 4 °C followed by flow cytometry analysis. The frequencies of IFN-γ⁺ cells in CD3⁺/CD8⁺ populations were calculated.