Accuri a6 cytometer

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⁺.

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 for 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 CD3 (Clone SK7), APC CD4 (Clone RPA-T4, BD Pharmingen) antibodies. Dendritic cell staining was performed on 238 cord blood samples using FITC Lin-2 (clone MφP9/SJ25C1/SK7/L27/NCAM16.2, anti-CD3, CD14, CD19, CD20, CD56), PE CD123 (clone 9F5), PerCP HLA-DR (clone L243), and APC CD11c (clone S-HCL-3, BD Pharmingen). Cells were immediately analyzed 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⁻, and plasmacytoid cells were defined as Lin-2⁻HLA-DR⁺CD11c⁻CD123⁺. To normalize the frequency of analyzed CD4 subsets (T_reg cells) from cryopreserved CBMCs to absolute rates of CD4 per microliter of fresh whole cord blood, absolute CD4 subset counts were calculated (CD4 subset frequency * absolute CD4 count per microliter of whole cord blood).