P35g 1.0 14 c

HeLa and COS-7 cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium/F-12 with GlutaMAX (ThermoFisher) supplemented with foetal bovine serum (FBS, 10%, Sigma). Cells were maintained at 37 °C in humidified air with 5% CO₂ and passaged every 3–4 days using Gibco TrypLE Express (ThermoFisher). For imaging, cells were grown on 35-mm glass-bottomed dishes (#P35G-1.0-14-C, MatTek) coated with human fibronectin (10 μg.ml⁻¹). Cells were transfected, according to the manufacturer’s instructions, using TransIT-LT1 (GeneFlow) (1 μg DNA per 2.5 μl reagent). Short tandem repeat profiling (Eurofins, Germany) was used to authenticate the identity of HeLa cells [7 (link)]. Screening confirmed that all cells were free of mycoplasma infection.

To observe the formation of autophagosomes in VW oocytes from young and aged mice, oocytes were stained with CYTO-ID Green dye (1:500, Enz-51031-0050, Enzo life sciences, Farmingdale, NY, USA) and Hoechst (1:1000, H33258, Sigma-Aldrich) at 37 °C in 5% CO₂ for 30 min. CYTO-ID Green dye, a cationic amphiphilic tracer dye, specifically labels autophagic compartment, indicating that the puncta stained with CYTO-ID are a specific autophagy marker [22 (link)]. The oocytes from young mice were incubated with 100 nM rapamycin (Enz-51031-0050, Enzo life sciences) at 37 °C in 5% CO₂ for 2 h as the control of autophagosome formation in oocytes before CYTO-ID Green staining. The oocytes were fixed, permeabilized, and then incubated with Phalloidin (1:100, A22287, Invitrogen) in PBS for 15 min. Oocytes were washed three times with PBS for 10 min each. After a final wash in PBS, the oocytes were directly placed onto a glass-bottom confocal dish (P35G-1.0-14-C, MatTek), observed using a confocal microscope (Zeiss LSM880, Carl Zeiss), and analyzed using ZEN software (Carl Zeiss).

To observe the mitochondrial distribution in oocytes before and after vitrification, live oocytes were washed two times with M2 media and stained with MitoTracker^TM Red CMXRos (200 nmol/L, M7512, Invitrogen) at 37 °C in 5% CO₂ for 15 min. They were then treated with NucBlue^TM Live Cell Stain ReadyProbes^TM reagent (R37605, Invitrogen) for 15 min in an incubator. Oocytes were washed two times with M2 media and transferred to a glass-bottom confocal dish (P35G-1.0-14-C, MatTek, Ashland, MA, USA). Live images of mitochondria in oocytes (red color) were obtained directly using a confocal microscope (Zeiss LSM880), and analyzed using ZEN software (ZEN 2012, Blue edition, Carl Zeiss) after red was converted to pseudo-color (green) for better visualization. For quantification of the mitochondrial distribution, fluorescence intensity profiles were measured using the ImageJ software (v1.48, NIH, Bethesda, Maryland, USA).

Embryos were cultured in glass-bottom dishes (MatTek Corporation; P35G-1.0-14-C) in a drop of KSOM (supplemented with sodium pyruvate, essential and nonessential amino acids) pre-equilibrated overnight at 37°C in 5% CO₂. Culture medium was overlaid with pre-equilibrated mineral oil (Sigma-Aldrich; M8410) to prevent evaporation. Embryos were imaged on a Zeiss LSM 710 confocal microscope equipped with an environmental control chamber, using a 40×1.2 NA water immersion objective. EGFP was excited at 488 nm and TdTomato at 560 nm. To prevent embryos from drifting out of the field of view, their movement was restricted using hand-pulled thin glass filaments secured with vacuum grease (supplementary material Fig. S1). We collected 20 focal planes spanning the entire embryo at 15 min intervals. Embryos were imaged for up to 30 h until they reached the blastocyst stage. The excitation laser power was kept at the lowest possible (560 nm at 0.3%; 488 nm at 0.2%) and images were taken at an in-plane resolution of 128×128 pixels. We empirically determined imaging parameters suitable for normal development (as assessed by transfer into recipients) by starting at high image quality levels (512×512×40 pixels, laser power of ∼3%) and then worked our way down to lower resolutions until we obtained satisfactory viability. We disabled image averaging to further reduce pixel dwell time.

One day before live cell imaging, HeLa cells were seeded in 35 mm petri dishes with 14 mm microwells (P35G-1.0-14-C MatTek). LysoSensor Yellow/Blue DND-160 (L-7545 Molecular Probes) probe was added at 1.5 μM in full medium for 20 min, removed, then the cells washed twice and visualized at a confocal microscope. Primary cortical neurons were seeded in the MatTek live-cell dishes, treated with shRNA lentiviral particles (standard protocol: 20 μl of the 100 × viruses stock on day 4, media changed in the following day and cultured for another 5–6 days) and probed for LysoSensor Yellow/Blue at 1.5 μM in full medium for 5–10 min. The fluorescence intensities in the blue and yellow channels were quantified in ImageJ.
The lysosomal protease activity was measured using the Cathepsin L Activity Kit (65306 Abcam) following the manufacturer's instructions.

The experimental procedures described in ref. ^{50 (link)} were adapted for ex vivo live imaging of midguts. Our ex vivo live-imaging medium comprises Schneider’s Drosophila medium (Thermo Fisher Scientific, 21720024), 2% FBS (Life Technologies, 16140071), and 0.5% penicillin-streptomycin (Thermo Fisher Scientific, 15140122).
Drosophila were dissected in the imaging media and the entire midgut and hindgut sections were recovered. Then, the dissected samples were mounted on a 35-mm glass bottom dish (MatTek, P35G-1.0-14-C). To prevent squeezing, vacuum grease was applied as two lines on the glass bottom dish, ~2 mm apart. Approximately 100 µl of imaging media was added to the center of the dish, and the dissected guts were placed perpendicular to the vacuum grease lines. Anterior end of the midgut and hindgut segment were embedded in each vacuum grease line to prevent drifting. A cover glass was gently placed on top of the vacuum grease lines to prevent excessive movement of the midgut. Approximately 1 ml of imaging medium was added to the dish to prevent dehydration and facilitate gas exchange. To prevent media evaporation, the samples were covered with the dish lid and were imaged from the bottom side.