Apc conjugated cd11c

For characterisation of LA-4 cells, the following antibodies were used: PE-conjugated anti-mouse TLR5 (clone: 85B152.5, 1:100, Abcam, Cambridge, UK), PE-conjugated anti-mouse MHCI (clone: 31-1-2S, 1:100, eBiosciences, Frankfurt, Germany), AF488-conjugated anti-mouse Pan-cytokeratin (clone: AE1/AE3, 1:50, eBiosciences), AF488-conjugated anti-human mucin 1 (clone: SM3, 1:25, eBiosciences), APC-conjugated anti-mouse EpCAM (clone: G8.8, 1:50, eBiosciences), and APC-conjugated anti-mouse CEACAM1 (clone: CC1, 1:50, eBiosciences). Bone marrow-derived mDCs were characterized using anti-mouse pacific blue-conjugated CD11b (clone: M1/70.15, 1:50, Invitrogen, ThermoFisher Scientific, Dreieich, Germany), APC-conjugated CD11c (clone: HL3, dilution: 1:500, BD Bioscience), and PE-Cy5-conjugated B220 (clone: RA3-6B2, dilution: 1:100, BD Bioscience), anti-mouse FITC-conjugated CD3 antibody (clone: 145-2C11, 1:50, BD Biosciences), anti-mouse FITC-conjugated CD19 antibody (clone: 6D5, 1:50, Southern Biotech), and anti-mouse FITC-conjugated CD49b pan NK cell antibody (clone: Dx5, 1:100, BioLegend). Cells were analyzed by flow cytometry using a LSR II flow cytometer (BD Bioscience). Data were analyzed using FlowJo either V.7 or V.10 (Treestar Inc., Ashland, OR, USA).

Dendritic cells and CD4 T cells were enumerated from 50 μL of whole cord blood stained with antibodies in BD TruCount™ tubes or with 20 μL of CountBright™ counting beads (ThermoFisher Scientific). Cells were incubated 20 min, and 900 μL of BD FACS lysis solution was added for 15 min. CD4 T cell staining was performed on 152 cord blood samples using PerCP-conjugated CD3, APC-conjugated CD4 antibodies. Dendritic cell staining was performed on 145 cord blood samples using FITC-conjugated Lin-2 (CD3, CD14, CD19, CD20, CD56), PE-conjugated CD123, PerCP-conjugated HLA-DR, and APC-conjugated CD11c (BD Pharmingen). Cells were immediately analysed on an Accuri A6 cytometer. Dendritic cells were defined as Lin-2⁻HLA-DR⁺, myeloid dendritic cells were defined as Lin-2⁻HLA-DR⁺CD11c⁺CD123⁻, plasmacytoid cells were defined as Lin-2⁻HLA-DR⁺CD11c⁻CD123⁺.