Fluo 4 am

The cardiomyocytes were plated on a 35 mm confocal dish loaded with 4 μM Fluo-4 AM (Yeasen, China) and incubated at 37 °C for 20 min in PBS (Servicebio, China) containing 0.04% Pluronic F-127 (Yeasen, China). PBS was changed to the Cardiomyocytes Maintenance Medium (Cellapy, China). Loaded samples were transferred under a TCS‐SP5‐RS confocal microscope (Leica, Germany). Laser emission at 488 nm was used for stimulation and emitted fluorescence at 530 nm was acquired. Samples were then stimulated with freshly prepared solution of caffeine (20 mM) and emitted fluorescence acquired to record transient alteration in cytosolic calcium levels.

As previously described by Ma et al. (24 (link)), the calcium imaging method was performed. Briefly, the differentiated ND7/23 cells were washed with FACS buffer containing 2 mM CaCl₂ for three times and then incubated with 5 μM Fluo-4 AM (Yeasen Biotechnology Co., Ltd, China) for 40 minutes at 37°C. Following this, cells were washed by FACS for another three times and then preserved in 200 μL FACS buffer. Time-lapse images were acquired by Olympus IX83-FV3000-OSR (Olympus Optical Co., Ltd, Tokyo, Japan) with excitation at 488 nm and emission at 500–600 nm. Before the addition of stimulators, seven baseline fluorescence readings were taken, followed by fluorescent readings every second for 300s. The ratio of real-time fluorescence divided by baseline fluorescence (F/F_base) was utilized for each well to normalize the Ca²⁺ signals.

Intracellular Ca²⁺ was measured by Fluo 4‐AM (40704ES50, Yeasen), a membrane‐permeable fluorescent indicator for Ca²⁺. Briefly, HT22 cells were washed three times with Ca²⁺ and Mg²⁺‐free Hanks' balanced salt solution (HBSS) and loaded with Fluo 4‐AM (4 μM) for 40 min at 37°C in HBSS. The cells were then washed three times with HBSS and imaged using a fluorescence microscope equipped with a CCD camera (Mshot).

Mouse platelets were preloaded with Fluo-4 AM (5 μM, Yeason, 40704ES50) for 30 min in Tyrode’s buffer without calcium and then left for another 30 min. After washing, platelets were stimulated with CRP (0.5 μg/ml) or thrombin (0.005 U/ml) in the presence of 2 mM CaCl₂ and the fluorescence intensity was detected using a microplate reader (Biotek Synergy H1) at Ex/Em = 490/525 nm. Calcium mobilization was quantified as the ratio of the fluorescence intensity after stimulation relative to that resting platelets.

Briefly, HepAD38 cells were seeded in 96-well plates at 2 × 10⁴ cells per well and cultured in DMEM/F12 for 24 h. Cells were then washed with HBSS buffer three times and incubated with 5 μM Fluo4-AM containing 0.05% PluronicF-127 (Yeasen Biotech, Co., Ltd.) at 37°C for 1 h. The cells were washed three times with HBSS and incubated at 37°C for another 30 min to make the fluorescence probe completely de-esterifying. The cells were then incubated with serially diluted compounds and the Ca²⁺ levels were determined based on the fluorescence (excitation wavelength 480 nm, emission wavelength 525 nm) measured using a Perkin Elmer Envision Multi-label Plate Reader.

CCD-Co18 cells were seeded in a 6-well plate and cultured till the confluency was ~50%. The medium was replaced with serum-free medium containing the isolated exosomes, and the cells were cultured for 48 h. The cells were then stained with 4µM Fluo-4 AM (40704ES50, Yeasen) and observed under a fluorescence microscope at the excitation wavelength 475 nm and emission wavelength 560 nm.