Synergy cell sorter

For PGC quantification, the entire embryo (E8.5-10.5) or the developing urogenital ridges (E11.5-13.5) were isolated from embryos and placed into 500 μL or 150 μL pre-warmed 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) respectively, and incubated at 37 °C for 10 min. Subsequently, 1 μL of Benzonase endonuclease (Millipore) was added and the sample gently disaggregated by pipetting and incubated for 5 min at 37 °C. The trypsin was inactivated by adding 1 mL of PBS/5% v/v fetal bovine serum and centrifuged at 1000 x g for 10 min. The sample was resuspended in 100 μL of anti-SSEA1 conjugated to Alexa Fluor 647 (catalog no. MC-480; Biolegend) diluted 1:100 in PBS/2.5% v/v fetal bovine serum and incubated for 10 min at room temperature. Samples were diluted by adding 300 μL of PBS/2.5% v/v fetal bovine serum and passed through a 70 μm filter. For quantification, 300 μL of the samples were immediately run on an ECLIPSE analyzer (Sony Biotechnology) and the data analyzed using FlowJo v10. For sorting of cells, samples were immediately run on a Synergy cell sorter (Sony Biotechnology), the cells sorted into 10 μL of PBS, centrifuged at 3500 x g for 5 min and stored at −80 °C until further analysis.

For flow cytometry analysis, up to 4 × 10⁶ DT40, HEK293T or U-2OS cells were harvested each time by centrifugation at 1000 rpm, 4°C for 10 min. For fluorescent-activated cell sorting (FACS), up to 5 × 10⁷ DT40 cells were harvested. The cells were washed once with PBS before staining with the appropriate antibodies and/or antigens. Surface human scFv/HEL expression was detected each time by staining with a mouse anti-HEL F10 antibody (a kind gift of Dr R. Poljak). All antibodies and antigens were diluted in PBS/1% BSA for staining. Antigens at final concentrations of 10–100 nm were used each time. Cells were stained for 20 min on ice, with washings in between each staining step using cold PBS. Stained cells were resuspended in cold PBS/1% BSA and analysed on either the FACS Calibur (Becton Dickinson) or the BD LSRII (Becton Dickinson). Flow cytometry plots were made using FlowJo Version 9. FACS was performed on either the MoFlo High Speed Cell Sorter (Propel Labs) or the Synergy Cell Sorter (Sony Biotechnology).