Y 27632

Cells were digested by 0.05% trypsin-EDTA, and the digested cells were filtered through a 40 μm cell strainer and centrifuged at 1200–1500 rpm for 3 min at room temperature. The supernatant was removed, and the cells were resuspended using culture medium with the addition of Y-27632 (10 μM; Tocris, 1254) and placed on the ice before injection. After being placed on ice, the digested cells should be injected after 1 h; otherwise, another batch of cells were digested for the remaining injections.
Single cells were microinjected into 8-cell ICR diploid mouse embryos. The injected embryos were cultured in the culture medium with Y-27632 (10 μM; Tocris, 1254) for the first 4 h. For the generation of chimeric blastocysts, the embryos were transferred into the KSOM medium (Merck, MR-106-D). For the generation of single-cell-derived in vivo chimeric conceptuses, chimeric embryos were cultured in KSOM medium with Y-27632 (10 μM; Tocris, 1254) addition in a humidified incubator under 5% CO₂ at 37 °C overnight. Injected embryos were transferred to uterine horns of 0.5 dpc pseudo-pregnant females. Fetal tissues, yolk sacs and placentas were dissected from conceptuses at E7.5, E10.5, E13.5 or E17.5 developmental stages.

To activate RhoA, primary RPE cells were incubated with cell-permeable exoenzyme RhoA activator that has been shown to activate Rho specifically [34 (link)]. RhoA activator (#CN03, Cytoskeleton Inc) was applied to RPE cells in culture at 1 μg/mL in DMEM 10% FBS for 3 h directly prior to POS phagocytosis assays. The widely used and well-characterized ROCK-specific competitive inhibitor Y-27632 (#1254, Tocris Bioscience, Minneapolis, MN, USA, [35 (link)]), was applied at 25 μM during POS challenge for cell culture assays. Initial experiments established similar effects on promoting POS internalization by wt RPE in the absence of MerTK ligand for Y-27632 at 50 μM, and efficacy of other ROCK inhibitors, H-1152 (#2414, Tocris Bioscience) and HA-1100 (#2415, Tocris Bioscience), used at 5 μM and 50 μM, respectively (Supplementary Figure S1).
To induce phagocytosis by RPE tissue ex vivo, posterior RCS rat eyecups dissected 5 min after light onset were incubated in DMEM with 10% FBS and with and without 50 μM Y-27632 for 2 h followed by labeling and immediate live imaging as described in Section 2.1.

The 34D6, male, human iPSC line (a gift from Prof Chandran, Edinburgh) 43 was cultured in mTeSR1 (Stem Cell Technologies) following the manufacturer's instructions. Tissue culture plates (Nunclon, Invitrogen) were coated for at least 2 hours with MatrigelTM (BD Biosciences, VWR) diluted 1:75 with DMEM/F12 (Invitrogen). 34D6 iPSCs were plated at a density of ~10⁶ cells/10‐cm plate in complete mTeSR1TM medium containing 10 μM Y27632 (Tocris) and incubated at 37°C in a standard 5% CO₂, humidified incubator (Binder). To passage iPSC, cells were first treated with 10 μM Y27632 for 2 h, then washed with Ca²⁺/ Mg²⁺‐free PBS and cell colonies lifted from the plate by incubation with Dispase (Stem Cell Technologies) containing 10 μM Y27632 (Tocris). Colonies were fragmented by gentle trituration, collected by centrifugation (180 g) and re‐suspended in medium for re‐plating.