*4.5. Flow Cytometry Analysis*

Human LNSCs (passages 5–10) were harvested from a 6-well dish using 1 mL TripLE™ Select (Gibco, Bleiswijk, The Netherlands) for 10 min at 37 ◦C. Subsequently, cells were washed in PBA buffer (PBS containing 0.01% NaN3 and 0.5% BSA (Sigma Aldrich, Zwijndrecht, The Netherlands), and stained for 1 h with rat IgG2a anti-human Podoplanin (clone NZ-1, AngioBio, Huissen, The Netherlands) on ice. Afterwards cells were washed again in PBS buffer, followed by a second incubation for 30 min on ice protected from light using the following directly labelled antibodies: polyclonal goat anti-rat IgG AlexaFluor647 (Invitrogen, Landsmeer, The Netherlands), CD45 FITC (clone HI30, Becton Dickinson (BD) Pharmingen, Vianen, The Netherlands), CD80 PE (clone 2D10.4, eBioscience, Landsmeer, The Netherlands), CD274 (PD-L1) BV421 (clone MIH1, BD Biosciences, Vianen, The Netherlands), HLA-DR PE-Cy7 (clone L243, Sony Biotechnology, Surrey, UK), and CD40 PE-Cy7 (clone 5C3, Sony Biotechnology, Surrey, UK) or with corresponding isotype control antibodies. Staining with HLA-ABC PE-Cy7 (clone G46–2.6, Biolegend, London, UK) served as a positive control and was used to set-up the correct compensation configuration settings. To assess the HLA-DR and co-stimulatory expression on human LNSCs directly ex vivo, freshly isolated cells were stained with eBioscience™ Fixable Viability Dye eFluor™ 780 (Invitrogen, Landsmeer, The Netherlands) for 15 min, followed by 10 min blocking in PBS containing 5% normal human serum and 2% FCS (Biowest) on ice in the dark. The cells were then incubated with CD45 eFlour 450 (MI30, Invitrogen, Landsmeer, The Netherlands), CD11c Alexa 700 (Bu15, Biolegend, London, UK), HLA-DR PE (clone L243, Invitrogen, Landsmeer, The Netherlands), CD80 Pe-Cy7 (clone 2D10, Biolegend, London, UK), CD86 Pe-Cy5 (clone IT2.2, Biolegend, London, UK), CD40 PerCP/Cy5.5 (clone 5C3, Biolegend, London, UK), PD-L1 BV711 (clone 29E.2A3, Biolegend, London, UK) antibodies for 30 min on ice in the dark. For staining of AIRE protein in LNSCs we used the Fixation/Permeabilization Solution Kit (BD Biosciences, Vianen, The Netherlands) to detect intranuclear expression or the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, Landsmeer, The Netherlands) according to the manufacturer's instructions. Cells were stained with AIRE PE (clone 614530, R&D Systems, Minneapolis, MN, USA) and mouse IgG1 isotype control (eBioscience, Landsmeer, The Netherlands) for 30 min at RT. Cells were measured on a FACS CANTO II (BD, Vianen, The Netherlands) or a BD LSRFortessa™ X-20 (BD Biosciences, Vianen, The Netherlands) and analyzed with FlowJo software (TreeStar Inc., Ashland, OR, USA).
