5 fluoro 2 deoxyuridine

To analyze autophagic flux, hPSCs with the OPTN(E50K) mutation as well as isogenic controls both lacking the BRN3b-tdTomato-Thy1.2 reporter were used. Differentiated retinal organoids were enzymatically dissociated with AccuMax and plated on 12 mm coverslips in Brainphys medium for 4 weeks, as described above. Dissociated cells were treated with 1 μM of 5-fluoro-2′-deoxyuridine (Sigma, cat. no. F0503) for the first 24 hours to remove presumptive progenitor and/or glial cells. The RFP-GFP-LC3B sensor (Life Technologies, cat. no. P36239) was added to hPSC-RGCs after 4 weeks of maturation for a duration of 16 hours. Subsequently, immunohistochemistry was performed to stain the cells with a BRN3 primary antibody to definitively identify RGCs, followed by an Alexa Fluor 647 anti-goat secondary antibody. Immunofluorescence images were visualized on a Nikon A1R Confocal Microscope with Z-stack. To ensure the proper selection of RGCs, only cells expressing BRN3 were considered for further analysis of RFP-GFP-LC3B expression. Quantification of autophagosomes (both RFP and GFP puncta) and autolysosomes (RFP positive, GFP negative puncta) was performed on RGC somas through Fiji by using JACop plugin with appropriate threshold. Autophagosomes were calculated as the fraction of RFP puncta overlapping with GFP puncta.

Primary cultures of mouse hippocampal neurons were prepared according to previously described techniques33 (link). In brief, 15-day-old embryonic C57BL/6 J mice were anesthetized with halothane. Brains were removed rapidly and placed in ice-cold Ca²⁺- and Mg²⁺-free phosphate buffered solution. Tissues were dissected and incubated with 0.05% trypsin-EDTA for 10 min at 37 °C, followed by trituration with fire-polished glass pipettes, and plated on poly-D-lysine-coated 35 mm culture dishes at a density of 1 × 10⁶ cells per dish. Neurons were cultured with Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen) and maintained at 37 °C in a humidified 5% CO₂ atmosphere incubator. Cultures were fed twice a week and used for electrophysiological recording 10–20 days after plating. For neuron cultures, glial growth was suppressed by addition of 5-fluoro-2-deoxyuridine (20 μg/ml; Sigma-Aldrich) and uridine (20 μg/ml; Sigma-Aldrich).
Human embryonic kidney (HEK)-293T cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. The cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 1 mM L-glutamine, 10% foetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin (all from Invitrogen).

5-Fluoro-2′-deoxyuridine (FUDR, ≥99.9%) and the ROS Green Fluorescence Detection Kit were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, United States). Methyl viologen dichloride (paraquat, 98%) was provided by Aladdin Reagent Co. (Shanghai, China). All other chemicals and solvents were of analytical grade or above.
The C.elegans strains and Escherichia coli OP50 used in the study were provided by Luo Huairong’s research team, Kunming Institute of Botany. The C.elegans strains included the wild-type C.elegans N2 (Bristol), TJ356 (daf-16:GFP), daf-16(mu86) I, daf-2(e1370), age-1(hx546), skn-1(zu135) IV,LD1 (skn-1:GFP), CL2166(gst-4:GFP), CF1553 (sod-3:GFP), hsf-1::GFP(OG532) strains. All strains were grown and maintained at 20°C on solid nematode growth medium (NGM) plates.Among them, the strains were all from LUO Huairong’s and Shao Zhiyong’s research teams.
Metabolite profiles were generated through ultrahigh-performance liquid chromatograph Q extractive mass spectrometry (UHPLC-QE-MS) by Shanghai Biotree Biomedical Technology Co., LTD.