Rhod 2 am

Cytosolic Ca2+ levels were determined using Fura-4 AM (Yeasen, Shanghai, China) and Rhod-2 AM (40776ES50, Yeasen, Shanghai, China). Cells were loaded with Fura-4 AM or Rhod-2 AM in HBSS for 30 mins at temperature of 37°C and then the solutions were removed. Then, cells were washed with HBSS for 3 times to fully remove Fura-4 AM or Rhod-2 AM, and the cells were covered with HBSS for 30 mins at temperature of 37°C. Fluorescence images were obtained using an OLYMPUS CKX5 fluorescence microscope (Olympus, Tokyo, Japan).

The rhodamine-2 acetoxymethyl ester (Rhod-2/AM) fluorescent probe was used to detect the mitochondrial calcium content as described before [13 (link)]. Briefly, SH-SY5Y cells were seeeded in 6-well plates and loaded with 5 μM Rhod-2 AM (Yeason, Shanghai, China) in Hank’s buffered salt solution (HBSS) for 30 min at 37 °C. After rinsing with PBS to remove excess or non-specific probes loaded in mitochondria, cells were incubated further for 30 min to allow complete de-esterification of intracellular AM esters. Then, cells were collected and gently resuspended in 200 μL PBS for flow cytometry. To determine the mtROS production, cells were loaded with 2.5 μM MitoSOX (Thermo Fisher Scientific, United States) for 10 min at 37 °C, washed with PBS, collected, and resuspended in PBS. The loaded cells were then analysed using a Sony ID7000 Spectral Analyser (Sony Biotechnology, Tokyo, Japan). MMP and ROS generation rate are expressed as the proportion (%) of cells with high fluorescence emission intensity.

The calcium concentrations of the cytoplasm, mitochondria and endoplasmic reticulum were measured using the calcium ion fluorescent probes, Fluo-4 AM, Rhod-2 AM and Fluo-5 N (Yeasen, Shanghai, China), respectively [21 (link), 22 (link)]. Working solutions of Fluo-4 AM, Rhod-2 AM and Fluo-5 N were prepared according to the kit instructions. The treated cells were washed three times with an HBSS solution (without calcium). Working solution (300 μL) was added to each well and incubated at 37 °C for 30 min. Cells were washed two times with 0.5 mL HBSS containing 2 μL EGTA, followed by 1 wash with the HBSS solution. Finally 300 μL HBSS solution was added to each well, incubated at 37 °C for 15 min. The fluorescence levels of cytoplasm Fluo-4 AM and endoplasmic reticulum Fluo-5 N were determined at Ex/Em = 494/516 nm. The fluorescence of mitochondria Rhod-2 AM was measured at Ex/Em = 516/549 nm and normalized based on cell protein concentration.

The content of Ca²⁺ in the cells was measured by fluorescent Ca²⁺ indicator Rhod-2 AM (#40776ES50, Yeasen, China). According to the manufacturer’s manual, ARPE-19 cells were infected with VZV for 72 h, and the medium was removed and the cells were rinsed with 1 × PBS at room temperature three times. After staining with 4 μM Rhod-2 AM for 30 min at room temperature (25°C), the fluorescent indicator was removed. The cells were rinsed with 1 × PBS at room temperature three times before observation and then placed in a 37°C, 5% carbon dioxide humidified incubator for 30 min. The images were taken by the laser confocal microscope (Olympus FV3000, Japan) at the excitation wavelength of 549/578 nm.

The fluorescent dye Rhod-2/AM (Yeasen, China) and Mito-Tracker Green (Beyotime, China) were used to measure primary myoblasts and C2C12 cells mitochondrial Ca²⁺ uptake, following the manufacturer’s instructions [51 ]. Briefly, primary myoblasts or C2C12 cells were incubated with Rhod-2/AM and Mito-Tracker Green to allow the cells to load the dyes according to the protocol. Then, the cells were permeabilized by 0.005% saponin (MedChemExpress, USA) for ~1 min, and the permeabilization solution was replaced with Ca²⁺-free HBSS solution. Cells were viewed with a spinning disk confocal super-resolution microscope (SpinSR10, Olympus, Tokyo, Japan) at the following wavelengths: 549 nm (excitation) and 578 nm (emission), and the frame rate was 2 frames/s. At the 15 s point, the Ca²⁺-free HBSS solution was replaced with 5 μmol/L CaCl₂ solution and continued to acquire images for an extra 50 s. Select the colocalization regions of Rhod-2/AM and Mito-Tracker Green to calculate the intensity of Rhod-2/AM by using Imaris software (Oxford, England) and ImageJ software. All the experiments were repeated three times.

4 kDa FITC-dextran was obtained from Maokang Biotechnology (Shanghai, China). Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco’s Modified Eagle Medium (DMEM) was procured from Gibco (USA). Antibodies for Piezo1, Occludin and ZO-1 were procured from Proteintech (China). The following chemicals were obtained from Servicebio (China): fluorescein (FITC) Tunel Cell Apoptosis Detection Kit, Phosphate Buffered Saline (PBS) and Hank’s Balanced Salt Solution (HBSS). Fluorescent-conjugated antibodies for flow cytometry were procured from BD Bioscience (USA). Secondary antibodies conjugated with AlexaFluor-488 or Alexa Fluor 594, Annexin V-FITC /PI Apoptosis Detection Kit, JC-1 Mitochondrial Membrane Potential Assay Kit, SYBR qPCR Master Mix, Rhod-2 AM and Fetal Bovine Serum (FBS) were procured from Yeason (China). Yoda1 and MitoSOx Red were procured from MedChemExpress (USA). MitoTEMPO was acquired from Topscience (China). DMEM no Ca²⁺ was obtained from Meiluncell (China).

HCE cells were carefully washed three times with Hank’s balanced salt solution (HBSS) and subsequently incubated with Rhod-2 AM (4 µM, Yeasen, Shanghai, China) for 60 min at 37 °C. Following the incubation, the cells underwent another set of three washes with HBSS and were further incubated for an additional 30 min at 37 °C. The intensities of Ca²⁺ were detected by flow cytometry (Beckman Cytoflex S, USA). FCM data were plotted and quantified with Flowjo software (Treestar).