Fortessa 3 flow cytometer

For analysis of maturation markers, cells were resuspended in ice-cold FACS buffer (5% FBS and 0.1% Na-azide in phosphate buffered saline (PBS)) containing diluted mouse serum (Agilent, Santa Clara, CA, USA), stained with BV421-conjugated anti-CD83 (BioLegend, San Diego, CA, USA), APC conjugated anti-CD86 (BioLegend) and PE-conjugated anti-HLA-DR (Leinco, St. Louis, MO, USA) antibodies for 30 min at 4 °C in the dark, washed with FACS buffer and subjected to flow cytometric acquisition. For viability analysis, cells were resuspended in Annexin V Binding Buffer (BioLegend) and stained for 15 min with PE-conjugated Annexin V (BD Biosciences, Franklin Lakes, NJ, USA) at 4 °C in the dark. PI (Biolegend) was subsequently added and the cells subjected to flow cytometric acquisition which was performed at the CFFC (Core Facility for Flow Cytometry, Faculty of Health and Medical Sciences, University of Copenhagen) using a Fortessa 3 flow cytometer (BD Biosciences). The acquired data were analyzed with FlowJo v10 software (Tree Star, Ashland, OR, USA).

Cells were pulsed for 30 min with 15 μM EDU and samples prepared using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Thermo Fisher #C10634) according to manufactures instructions. Total DNA was stained with FxCycle Violet Stain (DAPI) (Thermo Fisher Scientific, F10347). Cells were analyzed on a Fortessa 3 flow cytometer (BD). Doublets were gated out and 10,000 cells analyzed. The % of EDU positive cells, average EDU intensity and cell cycle distribution calculated using FlowJo software.