Fitc labeled goat anti mouse igg

Harvested cells were treated with Fc-receptor block (BD Biosciences, San Jose, CA) and incubated with either purified mAbs generated against rhAMHR2-ED or with isotype control mouse IgG. Cells were then treated with FITC-labeled goat anti-mouse IgG (BD Biosciences) and analyzed for antigen-specificity by flow cytometry using a FACSAria II flow cytometer and BDFacsDiva software (BD Biosciences). Positive control staining was performed using a commercially available mAb against AMHR2-ED (Abcam), whereas mouse IgG₁ isotype antibodies (Thermo Fisher) with irrelevant specificities were used as negative controls. Recombinant human AMH (LSBio, Seattle, WA) and recombinant ovalbumin (Sigma-Aldrich) were used in competitive binding assays.

NK-92 cells following 72 h transfection were harvested, washed with PBS and incubated with PE-labeled anti-NKp46 (BD Biosciences) for 30 min at 4 °C. After rinsed with PBS twice, cells were then incubated with FITC-labeled goat anti-mouse IgG (BD Biosciences) for another 30 min at 4 °C in the dark, and analyzed by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences).

HLA-A*0201 Ag-processing defective T2 cell line was cultured in RPMI 1640 containing 10% FBS. Before use, the cells were incubated for 6 h at 37 °C in serum-free IMDM. Then, cells were washed once, suspended in serum-free IMDM containing 20 μM of 2-ME and 15 μg/ml of human β2-microglobulin (β2m) (Calbiochem, La Jolla, CA), and pulsed with 50 μM peptide. HLA-A2.1-restricted MAGE-2 CTL epitope KMVELVHFL (amino acid position in MAGE-2; 112–120) and Kb-restricted Hpa CTL epitope FSYGFFVI (amino acid position in Hpa; 519–526) served as positive and negative controls. After a 24 h incubation at 37 °C, T2 cells were washed once with cold PBS containing 0.5% BSA and 0.02% NaN₃. They were then stained directly with primary anti-HLA-A2 Ab derived from BB7.2 and FITC-labeled goat-antimouse IgG (BD Biosciences Pharmingen, USA) secondary antibody. The percentage of FITC-positive cells as well as their staining intensity (mean fluorescence intensity (MFI)) was determined on an Epics Profile II (Coulter, Hialeah, FL). The Δ MFI for a particular mAb was calculated by subtracting the MFI of either the isotype-matched control mAb or the second-step Ab from each MFI value. The fluorescence ratio (FR) was calculated using the following formula: FR = (Δ MFI of peptide-treated T2 cells)/(Δ MFI of nontreated T2 cells).