M165 fc

Following isolation, all hearts were examined and photographed using a fluorescent stereo microscope (Leica M165 FC) to confirm plasmid expression. Plated cells were imaged using an Olympus FluoView FV1000 laser scanning confocal microscope. High-magnification in vivo imaging was performed by embedding cardiac tissue in 2% low melting temp agarose as described previously^{26 (link)}. Hearts were then imaged on a Zeiss 800 laser scanning confocal microscope. Calcium imaging using GCamp6F was conducted using a Hamamatsu ORCA-Flash4.0 CMOS camera at a rate of 100 fps at room temperature. The camera was mounted either on a Lecia M165 FC stereo microscope (Whole heart) or a Zeiss Axiovert S100TV inverted microscope using a 100× (NA 1.4) oil objective.

Lumbar spines from 2‐month‐old mice were dissected, followed by microdissection of NP and AF tissues using a fluorescent stereo microscope (Leica M165 FC). Isolated tissues were immediately fixed for RNA extractions. For primary cell culture (overview in Figure 1A), intact IVDs were dissected from 2‐month‐old mice (cervical to caudal) and the AF tissues were microdissected (Leica M165 FC). Isolated AF tissues were transferred to a sterile 3 mm culture dish with 2 mL of type II collagenase (3 mg/mL; Worthington, NJ) in Dulbecco's modified Eagle's medium/Ham's F‐12 medium (DMEM/F12) and incubated for 20 minutes at 37°C. AF tissues were then minced and further digested for 1 hour at 37°C. Digested tissues were triturated and filtered using a 70 μm cell strainer and cells were pelleted by centrifugation (1100 rpm for 5 minutes). Cells were plated at an initial density of ~400  000 cells/cm² and cultured in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (Thermo Fisher Scientific, MA) at 37°C in a humidified atmosphere of 5% CO₂. Media was changed every 2 days until cells reached 80% confluency. AF cells isolated from IVD tissues of two mice were pooled together and used for each experimental replicate.

Collagen gels were prepared in 96-well plates from a collagen solution (1.85 mg/mL, Cell Biolabs, Beijing, China) by following the manufacturer’s instructions. Briefly, HTM (4 × 10⁶ cells/mL medium) was added onto the collagen gel and incubated for 1 h at 37 °C with 5% CO₂. After collagen polymerization, the culture medium was added to each collagen gel lattice. Following a 48-h incubation, the edge of the gel was gently detached using a pipette tip. The gel area was then imaged using a Fluorescent Stereomicroscope (M165 FC, Leica) every hour for 15 h to determine the time required for cessation of the “natural contraction” of the gel by HTM cells. Drugs (NAC (3 mM) or VX-765 (100 μM)) were added to the medium, and images were captured at 6, 24, and 48 h. The gel area was calculated using the Fluorescent Stereomicroscope (M165 FC, Leica, China).