Quantifying Endogenous Sox10 in S16 Cells

For flow cytometric analysis of endogenous Sox10 levels, S16 cells were transfected with pCMV5-T7-Hes1 and pCAG-myc-Tle4-IRES-EGFP or the appropriate empty control plasmid using Xfect (TaKaRa Bio, Kusatsu, Japan), as described above. The next day, the medium was changed to the defined medium with 1 mM dibutyryl-cAMP and 5.7 µg/mL insulin and the cells were allowed to differentiate for 3 days. Subsequently, cells were detached using trypsin/EDTA, fixed with 4% PFA for 15 min in suspension, and permeabilized using PBS with 0.1% Triton X-100. Unspecific binding of antibodies was blocked with 10% FCS, 1% BSA, and mouse total IgG (dilution 1:100). Cells were stained with goat anti-Sox10 antiserum (dilution 1:200, self-made, RRID: AB_2891326, [36 (link)]), rabbit-anti GFP-AF488 antibody (dilution 1:200, A21311, Molecular Probes, Eugene, OR, USA), and mouse anti-T7 antibody (dilution 1:1000, Merck, Darmstadt, Germany). Secondary antibodies coupled to Cy3 and Cy5 were used (dilution 1:200, Dianova, Eching, Germany). The Sox10 median fluorescence intensity was subsequently analyzed on a LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lakes, NJ, USA) in only the transfected cell population (indicated by GFP and T7-Cy3 fluorescence, respectively). Data were analyzed with Flowing Software 2 (Turku Bioscience, Turku, Finland).