Journal: Current protocols in stem cell biology

Article Title: Differentiation Protocol for 3D Retinal Organoids, Immunostaining and Signal Quantitation.

doi: 10.1002/cpsc.120

Figure Lengend Snippet: Figure 2 Differentiation timeline for retinal organoids. The graphical overview of the differentiation protocol indicates the addition of different factors at specific time points and shows the development of the neural retina. The formed organoid at d1 develops a bright outer layer at d12 that resembles the neural retina. This layer becomes thicker over time. *d21: Transfer organoids to 48-well flat-bottom. M0, medium for organoid formation; M1, medium for retina formation; M2, medium for RPE formation; M3, medium for long-term culturing. R, Y- 27632 ROCK inhibitor; I, IWR1-endo; S, SB-431542; L, LDN-193189; CHIR, CHIR99021; SU, SU5402; RA, retinoic acid. Scale bar: 500 μm.

Article Snippet: DMEM/F12GlutaMAXTM Supplement 31331-093 ThermoFisher Scientific – – Y-27632 ROCK inhibitor S1049 Selleckchem 10 mM 20 μM Medium M0 see recipe in Reagents and Solutions (Table 8) AggreWell medium 5893 StemCell Technologies – – D-PBS (1×) 14190-169 ThermoFisher Scientific – – StemPro Accutase A11105-01 ThermoFisher Scientific – – IWR1-endo S7086 Selleckchem 10 mM 3 μM SB-431542 S1067 Selleckchem 20 mM 10 μM LDN-193189 S2618 Selleckchem 1 mM 100 nM Medium M1 see recipe in Reagents and Solutions (Table 8) Recombinant Human BMP4 314-BP/CF R&D Systems 50 μg/ml 55 ng/ml Medium M2 see recipe in Reagents and Solutions (Table 8) CHIR99021 S1263 Selleckchem 5 mM 3 μM SU5402 SML0443-5MG Sigma-Aldrich 10 mM 5 μM peqGOLD Universal Agarose 732-2789 VWR 1% 1% Water 1.153.332.500 Merck Millipore – – Medium M3 see recipe in Reagents and Solutions (Table 8) Retinoic acid R-2625-50 mg Sigma-Aldrich 100 μM 0.5 μM Table 2 Consumables used in Basic Protocol 1 Item Cat. no. Company Nunclon-Sphera 96-well U-bottom plate, sterile, with lid 174925 ThermoFisher Scientific 48-well flat-bottom suspension culture plate, sterile, with lid 677102 Greiner Reagent reservoir, sterile 4-0016 neoLab DISTRIMAN repetitive pipette F164001 Gilson DISTRITIP Maxi syringe tips, 12.5 ml F164150 Gilson 6.

Techniques: