Allophycocyanin conjugated streptavidin

The iPS clones were induced toward nephron progenitors using our previously described method^{17 (link), 18 (link)}, with a minor modification. At day 0–1, we added BMP4 at 0.25 ng/ml, instead of 0.5 ng/ml, to optimise the percentage of the ITGA8⁺/PDGFRA⁻ nephron progenitor fraction. Sorting of nephron progenitors was performed as described^{18 (link)}. The antibodies used for staining were as follows: phycoerythrin-conjugated anti-PDGFRA (Biolegend; 323506), biotinylated anti-ITGA8 (R&D Systems; BAF4076), and allophycocyanin-conjugated streptavidin (Biolegend; 405207). Data were obtained using a FACS SORPAria (BD Biosciences) and analysed with FlowJo software (TOMY Digital Biology).

rBC2LCN-FITC (180-02991; Wako), biotinylated anti-ITGA8 (BAF4076; R&D Systems, Minneapolis, MN, USA), allophycocyanin-conjugated streptavidin (405207; BioLegend, San Diego, CA, USA) and phycoerythrin-conjugated anti-PDGFRA (323506; BioLegend) were used for cell staining. Data were acquired and analysed using the MACSQuantify analysis software (Miltenyi Biotec, Bergisch Gladbach, Germany).

To evaluate the percentage of NPCs in day 12 NPC spheres, flow cytometry was performed. Biotinylated anti-ITGA8 (R&D Systems), allophycocyanin-conjugated streptavidin (BioLegend), and phycoerythrin-conjugated anti-PDGFRA (BioLegend) were used for cell staining. The allophycocyanin (Itga8)-positive and phycoerythrin (PDGFRA)-negative fraction was designated as the NPC population. The cut-offs were determined using a negative control without the primary antibodies. A minimum of 5 × 10⁵ cells was secured in each experiment.