Accuri 6c plus

Ficoll-isolated monocytes (500,000 cells) from fresh PB and BM samples (after the 1–2 days preculture) were stained with 1.25 µl allophycocyanin (APC)-conjugated anti-CD14 (BD Biosciences, Franklin Lakes, NJ; Cat. No. 561383), 5 µl phycoerythrin (PE)-conjugated anti-CD16 (BD Biosciences; Cat. No. 555407), and 1.25 µl fluorescein isothiocyanate (FITC)-conjugated anti-CD3 antibodies (Invitrogen; Cat. No. 11-0036) for 20 min at RT. Ficoll-isolated CB monocytes (500,000 cells) were stained with above-mentioned amounts of APC-conjugated anti-CD14, PE-conjugated anti-CD16 and 2.5 µl FITC-conjugated anti-CD45 antibodies (BD Biosciences; Cat. No. 555482) for 20 min at RT. Samples were run on the BD Accuri 6C Plus (BD Biosciences) flow cytometer and analyzed with FlowJo v10.7.1 software (FlowJo LLC.).

The following antibodies were used in the experiments: TruStain FcX anti-mouse CD16/32 (101320, BioLegend), FITC anti-mouse CD8 (A502-3B-E, ProImmune), Phycoerythrin (PE) anti-mouse CD3e (550353, BD Pharmingen), Peridinin-Chlorophyll-Protein (PerCP) anti-mouse CD19 (115531, BioLegend) and PE-Cyanine 7 anti-mouse CD4 (25-0041-82 eBioscience). SIINFEKL epitope-specific T cells were studied using APC-labeled H-2Kb/SIINFEKL pentamer (F093-84B-E, ProImmune). SVYDFFVWL (Trp2) epitope-specific T cells were studied using PE-labeled H-2Kb/SVYDFFVWL pentamer (F185-82B-E, Proimmune). SPSYVYHQF (AH1) epitope-specific T cells were studied using PE-labeled H-2Ld/SPSYVYHQF pentamer (F398-82A-E, Proimmune). Flow cytometric analysis were performed using a BD Accuri 6C Plus (BD Biosciences) or a BD LSRFortessa (BD Biosciences) flow cytometer and FlowJo software V.10 (BD Biosciences) was used for data analysis (see online supplemental figure 7 for gating strategies used in the experiments).