Flow cytometric immunophenotyping

Purity of IL-15 DC cultures was checked on a routine basis by multiparameter flow cytometry using a combination of FITC-, PE-, PB-/V450- and APC-conjugated monoclonal antibodies (mAbs) specific for CD3, CD7, CD11c, CD19 and CD56. All mAbs were from Becton Dickinson (BD; Erembodegem, Belgium) unless specified otherwise. Cell surface staining of mature IL-15 DCs was performed using FITC-, PE-, PB-/V450-, or APC-conjugated mAbs against various NK cell markers (CD7, CD16, CD56, CD69, NKG2D [Miltenyi], NKp46), NKDC-associated surface antigens (CD11c, B220 [eBioscience, Halle-Zoersel, Belgium], NKR-P1A/CD161 [Miltenyi]) and monocyte/DC markers (BDCA-1/CD1c [Miltenyi], CD14, CD40, CD80, CD83 [Invitrogen], CD86, CD209/DC-SIGN, CCR7 [R&D Systems, Minneapolis, MN, USA], HLA-DR). For detection of membrane-bound cytolytic effector molecules, DCs were stained with PE-labeled mAbs against TNF-α, Fas ligand/CD178 (eBioscience) and TNF-α-related apoptosis-inducing ligand (TRAIL). Dead cells were excluded by 7-amino actinomycin D (7-AAD; 5 µL/sample; BD) staining 10 min prior to acquisition on a FACSAria II (BD) flow cytometer. In each experiment, isotype-matched control mAbs were included to determine non-specific background staining. Results were expressed as delta mean fluorescence intensity (ΔMFI) (calculated by subtracting MFI values of isotype controls from sample MFI values) and as percentages of marker-positive cells (determined by Overton subtraction of isotype control histograms from sample histograms).