Glass bottomed dishes

The cells were seeded on glass-bottomed dishes (Matsunami Glass). After plasmid transfection, the cells were washed with PBS and fixed with 4% paraformaldehyde. After blocking with PBS containing 5% skim milk for 1 h at RT, the cells were incubated with anti-SARS-CoV-2 spike proteins (BLSN-005P, Beta Lifescience) or control IgG (Thermo Fisher) at 4 °C overnight. The following day, the cells were washed twice with chilled PBS and incubated with a secondary antibody labelled with Alexa Fluor 488 (Molecular Probes) for 1 h at RT. Cell nuclei were stained with DAPI (Roche). The cells were then observed under a confocal microscope (FV10i, Olympus).

The NRVMs were placed into glass-bottomed dishes (Matsunami Glass, Japan), and 200 nM Mito Tracker Green FM (MitoGreen) (Life Technologies, M7514) and 200 nM Mito Tracker Red CM-H2Xros (MitoRed) (Life Technologies, M7513) were added to each well. The NRVMs were incubated for 45 min at room temperature in the dark. After staining, the cells were observed under a digital microscope (Keyence, Japan).

Fura-2 imaging was performed as previously described with minor modifications (Tsuchiya et al, 2018 (link)). For indicator loading, MuSCs were plated on glass-bottomed dishes (Matsunami) coated with Matrigel and incubated with 5 μM Fura-2 AM (Dojindo) at 37°C for 60 min. Time-lapse images were obtained every 2 s. The base composition of HBS was (in mM) 107 NaCl, 6 KCl, 1.2 MgCl₂, 11.5 glucose, and 20 HEPES (pH = 7.4 adjusted with NaOH). HBS with 2 mM Ca²⁺ (2Ca) in addition contained 2 mM CaCl₂, whereas that without Ca²⁺ (0Ca) in addition contained 0.5 mM EGTA instead of CaCl₂. Ratiometric images (F₃₄₀/F₃₈₀) were analysed using Physiology software (Zeiss). Yoda1-induced Ca²⁺ influx was measured as the difference in the Fura-2 ratio between its maximum value and that at 1 min from the initiation of imaging. These experiments were performed using a heat chamber (Zeiss) to maintain the temperature at 37°C throughout the imaging process. The amplitude was calculated using the following formula: $\text{Amplitude}=\left(\left[{\text{maximum} \text{value} \text{of} \text{the} \mathrm{F}}_{340}{/\mathrm{F}}_{380} \text{ratio}\right]-\left[{\text{minimum} \text{value} \text{of} \text{the} \mathrm{F}}_{340}{/\mathrm{F}}_{380} \text{ratio}\right]\right)/2.$

Freshly isolated MuSCs were plated on glass-bottomed dishes (Matsunami) coated with Matrigel and immediately fixed with 4% PFA for 25 min. The MuSCs were then washed with PBS twice and permeabilised with 0.5% Triton X100/PBS for 10 min. They were blocked in 5% FBS/PBS and incubated for 1 h at room temperature with GST tagged Rhotekin-Rho binding domain (Cytoskeleton Inc.). After washing with PBS twice, cells were incubated in anti-GST Alexa Fluor 488 (1:500 dilution; Invitrogen) and DAPI in 5% FBS/PBS for 1 h at room temperature, before imaging.