Anti hla abc apc

Purity and phenotype of nDCs after immunomagnetic isolation were determined by flow cytometry with a FACSVerse® (BD biosciences, San Jose, CA) or MACS Quant® (Miltenyi Biotec). For this purpose, the following primary monoclonal antibodies and the appropriate isotype or fluorescence minus one controls were used: anti-CD1c-Viobright-FITC, anti-BDCA2-PE, anti-CD123-APC, anti-CD20-PE-Vio770, anti-CD45-APC-Vio770, anti-CD14-Viogreen, anti-FcεRI-BioBlue, anti-CD14-FITC, anti-CD15-PE, anti-CD56-APC, anti-CD3-VioBlue, anti-HLA-ABC-APC, anti-HLA-DR/DP/DQ-APC, anti-CCR7-APC, anti-CD80-APC, anti-CD83-APC, and anti-CD86-APC (all Miltenyi Biotec). Details are depicted in Supplementary Table 2. The purity of the nDC product was defined as the percentage of nDCs (sum of CD123⁺BDCA2⁺ pDC plus CD1c⁺CD20^- cDC2) of all viable cells in the nDC product. After 6 h of protamine/mRNA stimulation, cytokine production of nDCs was measured in the supernatant by cytometric bead array according to the manufacturer’s instruction (Miltenyi Biotec).

For flow cytometry analysis, cells were harvested, washed and re-suspended in 100 μl of PBS (Gibco) supplemented with 1% FBS (Gibco). Antibodies were added and incubated at 4 °C for 20 min in the dark. Samples were then washed and analysed by a FACSCalibur flow cytometer (BD Biosciences). To examine CAR expression, cells were first stained with biotin-SP (long spacer) AffiniPure F(ab’)2 fragment goat anti-mouse IgG (115-066-072; Jackson Immunoresearch Laboratories, Bar Harbor, Maine) followed by allophycocyanin (APC)-conjugated streptavidin (016-130-084; Jackson). For phenotyping of NK cells, the following anti-human antigen fluorescent conjugated antibodies were used: anti-CD56-APC (BD 555518, BD Biosciences), anti-CD45-PC7 (BD 557748, BD Biosciences), anti-CD3-PE (130-091-374, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-NKp46 (CD335)-PE (BD 557991, BD Biosciences), anti-NKp30 (CD337)-PE (BD 558407, BD Biosciences), anti-NKp44 (CD336)-PE (BD 558563, BD Biosciences), anti-NKG2D (CD314)-PE (BD 557940, BD Biosciences), anti-NKG2A(CD159a)-PE (IM3291U, Beckman Coulter, Brea, CA) and anti-CD94 (Kp43)-PE (IM2276, Beckman Coulter). For antigen detection, anti-CD326 (EpCAM)-APC (130-091-254, Miltenyi Biotec) and anti-HLA-ABC-APC (130-101-467, Miltenyi Biotec) were used.