Phase contrast microscope

The upper compartments of 6-well transwell plates were pre-seeded with rat retinal cells isolated as described (Supplemental Method 2) and maintained in neural stem cell culture medium. The lower chambers were then seeded with hPBMCs in suspension at 0.5 mL/well, and the plates placed in an incubator maintained at 37 °C with an atmosphere of 5% CO₂ and 100% humidity for 4 days. Each day, changes in hPBMCs morphology were examined under a phase-contrast microscope (Leica, Wetzlar, Germany). A half volume of neural stem cell culture medium in the upper compartment was exchanged for fresh medium each day.

ADSCs were observed by a phase-contrast microscope (Leica, German). And the ultrastructure was observed by transmission electron microscope (JEM-1400, Japan). Cell surface area was calculated with ImageJ (version 2.0.0, MD, USA). Cells were thresholded to remove the background to identify the whole-cell region of interest, and the area of each cell was measured and plotted.

A scratch wound healing model was conducted to check the migratory ability of PC3 cells following the transfection treatment. PC3 cells were plated at a density of 1 × 105 cells in 2-well Lab-Tek Chamber Slide (Sigma-Aldrich). After overnight incubation, cells were transfected with mimics or miR-NC. Wounds were created in confluent cells using a 200 μL pipette tip. The cells were rinsed several times with media to remove any free-floating cells or debris and the speed of wound closure was monitored after 24 h by measuring the change in distance of the wound edges. Each experiment was conducted in triplicate and representative scrape lines were photographed using a phase contrast microscope (Leica). For the invasion assays, after 24 h transfection, 1 × 105 cells in serum-free media were seeded onto the transwell migration chambers (8 μm pore size, Millipore). The insert in the upper chamber was coated overnight with 1 mg·mL⁻¹ BD Matrigel Matrix (BD Biosciences, Milan, Italy). A medium containing 10% FBS was added to the lower chamber. After 24 h, the non-invading cells were removed with a cotton-tipped swab and cells at the bottom of the Matrigel were stained with May–Grunewald–Giemsa stain (Sigma-Aldrich). Stained cells were counted under a microscope (Leica) at 200X magnification in 5 random fields in each well. Experiments were independently repeated three times.

Primary glial cells were seeded into the bottom chamber of a 24-well transwell plate with Matrigel Matrix-coated 3 μM pore size inserts (Corning) and subjected to OGD for 6 h with or without ASIV treatment. Then, purified NK cells (2×10⁵) were seeded into the upper chamber containing RPMI 1640 with 10% FBS at 37°C for 4 and 12 h. The fluid at 5 randomly selected fields in the lower chamber was collected in each well and the number of NK cells that migrated through the insert was counted under a phase contrast microscope (Leica, Germany).

The testicular tissues from the NOA-affected probands were fixed in 4% paraformaldehyde solution overnight, embedded in paraffin and sectioned at 5 μm thickness. Paraffin sections were dewaxed with xylene for 30 min, and then, rehydrated sequentially in 95, 90, 85 and 70% ethanol each for 10 min, followed in PBS solution for 10 min, and stained with hematoxylin and eosin according to standard protocols (catalogue number: ab245880, Abcam, Cambridge, UK). All the staining sections were captured by phase-contrast microscope to observe the structure changes of testicular tissues (Leica).