SSEA-1 Expression Analysis in Embryonic Urogenital Ridges

Quantification was performed as described previously^{62 (link)}. Urogenital ridges of E12.5 embryos were isolated and placed into 150 μl of trypsin solution (2.5 μg ml⁻¹ trypsin (Gibco), 25 mM Tris, 120 mM NaCl, 25 mM KCl, 25 mM KH₂PO₄, 25 mM glucose, 25 mM EDTA, pH 7.6) and incubated for 10 min at 37 °C. Next, 1 μl of Benzonase (Millipore) was added, followed by disaggregation of the sample by pipetting and incubation for for 5 min at 37 °C. trypsin was inactivated by adding 1 ml of PBS/5% v/v FBS. Following 10 min of centrifugation at 1,000g, the cell pellet was resuspended in 100 μl of Alexa Fluor 647-conjugated anti-human/mouse SSEA-1 antibody (catalog no. MC-480; BioLegend) diluted 1:100 in staining buffer (PBS/2.5% v/v FBS) and incubated at room temperature for 10 min; 300 μl of staining buffer were added to the cell suspension and samples immediately run on an ECLIPSE analyzer (Sony Biotechnology) and data analyzed using FlowJo v.10.1r5 (FlowJo LLC).