Ti e system

NK cell and TLNK spheroid images were obtained using an optical microscope (Ti-E System, Nikon, Tokyo, Japan). To assess clustering efficiency (i.e., spheroid volume and circularity), characterization was conducted using ImageJ software 1.53e. The spheroid volume was calculated using Formula (2): $$\mathrm{V}=0.5 \times \mathrm{Length} \times {\mathrm{Width}}^2$$
and circularity was calculated using Formula (3): $$\mathrm{Circularity}=4\pi \times \frac{\mathrm{Area}}{{\mathrm{Perimeter}}^2}$$

Alcian blue staining was also used to visualize cartilage ECM deposition by staining GAG contents in chondrogenically differentiated cells. After washing cells with PBS twice, cells were fixed using 4% pafaformaldehyde (Sigma-aldrich) for 30 min. After washing the samples 3 times to remove any remaining reagents, alcian blue solution (pH 2.5, Sigma-aldrich) was added and incubated for 1 h at room temperature. After a removal of staining reagents from each well, images of stained ADSCs were obtained using a camera-equipped optical microscope (Ti-E System, Nikon, Japan) [32 (link)].

Whole-cell patch recordings were performed at room temperature (23–26 °C) as we described previously^{36 (link),46 (link)}. Briefly, signal was amplified with an Axopatch 200B patch clamp amplifier connected to a digitizer Digidata 1440 and controlled with pClamp software 10 (Molecular Devices). A 1-s ramp voltage protocol from –100 mV to + 100 mV was applied at a frequency of 0.2 Hz. Signals were sampled at 10 kHz and filtered at 3 kHz. The buffered pipette solution contained (mM): 145 Cs-methanesulfonate, 10 BAPTA, 10 HEPES, and 8 MgCl₂, and the pH = 7.2 adjusted using CsOH. The standard bath solution contained (mM): 120 NaCl, 2.8 KCl, 10 CsCl, 2 MgCl₂, 10 CaCl₂, 10 HEPES, and 8 D-Glucose. The pH was adjusted to 7.4 with 1 M NaOH. The recording chamber had a volume of 150 µl and was perfused at a rate of about 2 ml min⁻¹. Thapsigargin (1 µM) was added in both pipette and bath solution to deplete the endoplasmic reticulum calcium store.
For Ca²⁺ imaging experiments, HK-2 cells were seeded on coverslips for 24 h and incubated with Fura-PE3 AM (1 μM) for 30 min at 37 °C in Ca²⁺ free standard bath solution. The ratio (F₃₄₀/F₃₈₀) of Ca²⁺ dye fluorescence was measured by a Nikon Ti-E system with NIS-Elements software as we reported previously^{16 (link),36 (link)}. All the experiments were performed at room temperature.

Organoids were cultured as described previously [18 ] in 8 μl BME (Cultrex) drops (70 basal and 60 luminal progenitor cells per drop) on nontreated 24 well plates and covered by Advanced DMEM/F12 supplemented with growth factors excluding Wtn3a or FGF2. Rock inhibitor (Y-27632) was added to culture medium for the first 4 days, and the medium was refreshed every 2–3 days. To induce R26creERT2-mediated deletion, 0.1 μM 4OHT was added to culture medium for 16–20 hours on day 1 or day 4 of culture. Organoids were photographed using best focus projection images on the Nikon TiE System software after 12–14 days in culture. Prior to proliferation measurements, RNA-seq, or ATAC-seq analysis, 12–16 day old organoids were dissociated into single cells using TrypLE express (Thermo Fisher Scientific). Cell suspensions were mixed 1:1 with CellTiter-Glo Luminescent Cell Viability Assay (Promega) prior to detection of luminescence. Genomic DNA was extracted from organoids using DNeasy Blood and Tissue kit (Qiagen) and PCR used to distinguish the Wt, floxed, and recombined (deleted, referred to as “del”) Suz12 alleles as described [34 (link)].

GMCs were treated with 30 mmol/L glucose alone or were intervened by 5 mmol/L lactisole following 30 mmol/L glucose for 24 h, respectively. For Ca²⁺ imaging experiments, GMCs on coverslips were incubated with Fura-PE3 AM (1 μM) for 30 min at 37°C in Ca²⁺-free standard bath solution. The ratio (F₃₄₀/F₃₈₀) of Ca²⁺ dye fluorescence was measured by a Nikon Ti-E system with NIS-Elements software. All the experiments were performed at room temperature (23–26°C).