Journal: bioRxiv
Article Title: Development of a protocol utilizing single-cell analysis for the differentiation of human iPSCs into SOX6+ midbrain dopaminergic neurons
doi: 10.1101/2025.06.18.660098
Figure Lengend Snippet: (A) Schematic depiction of the protocol for differentiating mDA neurons, illustrating the application of pharmacological compounds and growth factors. Representative immunocytochemical images and quantification from both original protocol and new protocol showing (B, E) efficacies of midbrain floorplate patterning at DIV11, showing co-localizations of LMX1+/EN1+ and SOX6+/OTX2+; and EN1+/SOX6+/TH+ neurons at DIV 16 (C, F) and 28 (D, G). White scale bars = 50, 100 µm as indicated (n = 3 independent experiments, error bars are SEM, ns = not significant; * = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001). (H) Calcium imaging recordings of spontaneous and KCl-induced activities on DIV 28 and 42, presenting the total number of calcium events per neuron recorded, and the percentages of cells responding to KCl-induced depolarization. Metrics of activities, including amplitude, rise time, and fall time of calcium signals, are depicted in the subsequent bar plots.
Article Snippet: The primary antibodies were used as follows: CORIN (rat, 1:1,000, R&D Systems, MAB2209), DCX (goat, 1:500, SantaCruz, sc-8066), EN1 (mouse 1:50, DSHB, 4G11), FOXA2 (goat, 1:500, R&D Systems, AF2400), GIRK2 (rabbit, 1:400, Alomone, APC006), LMX1A (rabbit, 1:4,000, Millipore, AB10533), LMO3 (goat, 1:200, SantaCruz, sc-82647), MAP2 (mouse 1:1,000, Sigma, M4403), NURR1 (rabbit, 1:500, SantaCruz, sc-990), OTX2 (goat, 1:1,000, R&D Systems, AF1979), TH (rabbit, 1:1,000, Millipore, AB152), TH (mouse, 1:500, ImmunoStar, 22941), and TH (sheep, 1:500, Novus, NB300).
Techniques: Imaging