Anti βiii tubulin tuj1

Cells were fixed with cooled 4% paraformaldehyde (PFA) for 15min and washed in PBS (0.1M). The cells were then blocked [PBS-0.25% Triton X-100+ 1% BSA (bovine serum albumin)] for 1h at RT and then primary antibodies (anti‐βIII tubulin (Tuj1, 1:1000, BioLegend), anti-NFH (1:1000, Merck), anti-CCR7 (1:200, Abcam; ab32527 (30 (link)))), were incubated O/N at 4°C in the same solution. After several washes, Alexa-Fluor-conjugated secondary antibodies were incubated in blocking solution for 1h at RT and cell nuclei were counterstained with Hoechst.

Cells were fixed with ice-cold 4% paraformaldehyde (PFA) for 15min and washed in PBS (0.1M). Blocking solution (PBS-0.25% Triton X-100+ 1% BSA (bovine serum albumin)) was then added and incubated for 1h at RT and then primary antibody (anti-βIII tubulin (Tuj1, 1:1000, BioLegend)), was incubated O/N at 4°C in the same solution. After several washes, Alexa Fluor 568-conjugated secondary antibody was incubated in blocking solution for 1h at RT.

iPSC‐derived neurons were fixed in 4% PFA, permeabilized in 0.3% TritonX‐100 in 2% bovine serum albumin, 3% goat serum containing phosphate‐buffered saline, and incubated with primary antibodies and fluorescently labeled secondary antibodies. The following primary antibodies were used: anti‐Tom20 (FL145, rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), anti‐TH (mouse monoclonal; Millipore, Billerica, MA), anti‐βIII tubulin/TUJ1 (mouse monoclonal, BioLegend; rabbit polyclonal from Abcam [Cambridge, MA]), anti‐FOXA2 (A12 clone, Santa Cruz Biotech). Images were obtained using UltraVIEW VoX Spinning Disk Confocal Microscope (PerkinElmer, Waltham, MA) and analyzed using CellProfiler. For dopaminergic makers, 30 images (63 × magnification) were analyzed for each marker per differentiation per line. For fragmentation and mitotracker measurements, 20 to 30 images (at 63×) were obtained per time point per clone per differentiation and >900 cells were analyzed per subject. For cell numbers, at least 20 images (20 × magnification) were obtained per time point per differentiation using immunofluorescence microscopy (696 × 520). Larger clusters that could not be quantified were excluded by the software, and, in total, >30,000 cells were counted per subject for cell death measurements.