Alexaflour647

Anti-CD4, anti-CD8, anti-CD11c, anti-CD11b, anti-MHC-II, anti-CD80, anti-CD86, anti-CD335, anti-CD3, anti-IFN-γ (all BD Biosciences, Heidelberg, Germany), anti-CD62L, anti-TNF-α, anti-Foxp3 (all eBioscience, Frankfurt, Germany), anti-CD160, anti-CD138, and anti-IL-10 (Biolegend, London, UK) were used as fluorescein isothiocyanate, pacific blue, phycoerythrin (PE), BD Horizon V450, allophycocyanin, AlexaFlour647, PE-cyanin 7, or peridinin-chlorophyll protein conjugates. Dead cells were identified by staining with the fixable viability dye eFlour 780 (eBioscience, Frankfurt, Germany). Intracelluar staining for Foxp3 was performed with the Foxp3 staining kit (eBioscience, Frankfurt, Germany) according to the manufacturer’s recommendations. Cytokine production from freshly isolated splenocytes was measured by stimulating cells with 10 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, München, Germany) and 100 µg/ml ionomycin (Sigma-Aldrich, München, Germany) for 4 or 6 h (IL-10), respectively, in the presence of 5 µg/ml Brefeldin A (for IFN-γ, TNF-α staining) and 5 µg/ml Monensin (for IL-10 staining), treating with 2% paraformaldehyde and 0.1% NP40, and staining with the respective antibody cocktail. Flow cytometric expression analyses were performed with an LSR II instrument using DIVA software (BD Biosciences, Heidelberg, Germany).

Mouse cells were dissociated from tissue culture plastic using a 1:4 mixture of Versene:Trypsin (0.05%) and human cells were dissociated using a 1:10 mixture of Cell dissociation buffer (Gibco 13150–016): Trypsin (0.05%). 5x10⁶ mouse or human cells infected with Notch3IC or Control overexpression vectors were stained with 0.2μg of either hamster anti-rat/mouse CD49a or isotype control conjugated to Alexa Flour 647 (BD Pharmingen 562113, and 562112) in 100μL of buffer (PBS with 0.1%NaN₃ and 0.5% bovine serum albumin). Cells were washed, fixed with 1% paraformaldehyde in buffer, filtered through a 35μm nylon mesh, and analyzed on a Gallios flow cytometer (Beckman Coulter). FlowJo 10.2 was used for gating and analysis. The number of replicates for each experiment are indicated in each figure legend.