Fitc conjugated anti ifn γ clone 4s b3

At the same time as phenotype detection, expression of intracellular IFN-γ, tumor necrosis factor (TNF)-α, CD107a or Granzyme B was determined by ICS. Total leukocytes were obtained from whole blood by lysis of erythrocytes using PBS containing 0.85% NH₄Cl. The cells were adjusted to ~5×10⁶/mL in RPMI 1640 culture medium supplemented with 10% FCS and stimulated with 100 ng/mL phorbol myristate acetate (PMA) plus 1 μg/mL ionomycin for 4 hours, in the presence of the secretion inhibitor monensin (BD Biosciences). Cells were stained with APC-conjugated anti-TCR γδ mAb, followed by washing with PBS, and fixation in 4% paraformaldehyde. Stained cells were permeabilized using 0.1% saponin (Sigma, St. Louis, MO, USA). Cells were incubated with FITC-conjugated anti-IFN-γ (clone 4S.B3; eBioscience, San Diego, CA, USA), PE-conjugated anti-TNF-α (clone Mab11; BD Biosciences), PE-conjugated anti-CD107a (clone H4A3; BD Biosciences), or FITC-conjugated anti-Granzyme B (clone GB11; BD Biosciences) for 30 minutes at 4°C. Finally, cells were washed with PBS, and analyzed using the FACS Canto II flow cytometer (BD Immunocytometry Systems). Data were analyzed using FACSDiva 2.0 software (BD Immunocytometry Systems).

After stimulation, cells were washed in wash buffer [PBS, 5% Fetal Bovine Serum (FBS), 0.1% sodium azide (Merk, Germany)] and stained with Krome Orange-conjugated anti-CD3 (clone UCHT1), Pacific blue-conjugated anti-CD8 (clone B9.11), PC-7-conjugated anti-CD279 (PD-1) (clone PD-1) and PE-conjugated anti-CD134 (clone Ber-ACT35) for 15 min at room temperature. All antibodies were purchased from Beckman Coulter, Krefeld, Germany.
Cells were fixed with 100 μl Reagent A (Caltag Labs., An der Grab, Austria) for 10 min. After washing, the pellet was resuspended in 100 μl permeabilization Reagent B (Caltag Labs.) and labeled with efluor660-conjugated anti-IL21 (clone eBio3A3-N2) or with efluor660-conjugated anti-IL22 (clone 22URTI) in addition to FITC-conjugated anti-IFN-γ (clone 4S.B3), all purchased from eBioscience, for 20 min in the dark. After staining, the cells were washed and immediately analyzed on FACS-Navios flow cytometer (Beckman Coulter).