Immortalized mouse cDC1 cell activation and analysis

Immortalized mouse cDC1 cells (22 (link)) were cultured in IMDM medium supplemented with 10% heat-inactivated FBS, 100x Glutamax, 10 mM HEPES, 100 μM Pen/Strep, 50 μM β-mercaptoethanol at 37° and 5% CO2. PBS supplemented with 20 mM HEPES and 5 mM EDTA was used for cell passaging. For activation, 2.5 x 10⁵ cells were seeded per well in a 12-well plate and incubated for 24 hrs with 5 μg/ml LPS and 10 ng/ml IFN-γ (485-MI, R&D Systems). Where indicated, cells were also incubated with 50 ng/ml recombinant Activin-A, TGF-β (Invitrogen), or BMP4 (Biotechne), or with 10 μM SB-431542. During the last 3 hrs of incubation, Golgi Plug (BD 555029) was added to the cells. Cells were then detached using PBS supplemented with 1% FBS and 2 mM EDTA, washed and stained for surface markers H2Kb FITC (Biolegend, clone AF88.5) and IA/IE AlexaFluor700 (BioLegend, clone M5/114.15.2), followed by fixation and permeabilization using FoxP3 staining buffer set (eBioscience) before intracellular staining with Ki67 eFluor 450 (Invitrogen, clone SolA15) and CXCL9 AlexaFluor647 (eBioscience, clone MIG-2F5.5). After the staining, cells were collected in FACS buffer (2% FBS, 2 mM EDTA in PBS), and data were acquired using an LSRII SORP or an LSR Fortessa cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).