Fluoview fv10

The levels of nuclear condensation and fragmentation were observed by means of nucleic acid staining with DAPI. The HT29 and HCT116 cells treated with plasma were harvested by trypsinization and fixed in 100% methanol at room temperature for 20 min. The cells were seeded on slides, stained with DAPI (2 μg/mL), and monitored by means of confocal laser microscopy (FluoView FV10, Olympus, Tokyo, Japan).

The proximity ligation assay (PLA) was performed using a Duolink in situ PLA kit (Sigma-Aldrich), according to the manufacturer’s procedure. In brief, cells (2 × 10⁵) were plated in a glass base dish (Iwaki/Asahi Glass Co., Funahashi, Japan) pre-coated with 0.01% poly-L-lysine (Sigma-Aldrich), and incubated for 24 h in DMEM supplemented with 7.5% FBS. After washing with cold PBS, the cells were fixed with 4% paraformaldehyde in PBS for 10 min at RT. Then, the cells were blocked with 10% donkey serum in PBS for 1 h, and incubated with mouse anti-GD2 mAb (220-51) and rabbit anti-integrin β1 Ab in 0.5% donkey serum albumin in PBS for 1 h at RT. The cells were washed twice with 0.05% Tween-20, and incubated with the Duolink in situ PLA probes anti-mouse PLUS and anti-rabbit MINUS for 1 h at 37 °C. After washing twice with buffer A (0.01 M Tris, pH 7.4, 0.15 M NaCl, and 0.05% Tween 20), a ligation solution was added and incubated for 30 min at 37 °C. Amplification was carried out using amplification reagent-polymerase solution for over 100 min at 37 °C. The samples were dried for approximately 10 min at room temperature in the dark, and mounted with a minimal volume of ProLong Gold antifade reagent with DAPI. The cells were analyzed under a confocal microscope (Fluoview FV10; Olympus, Tokyo, Japan).

To visualize T-tubules, isolated cardiomyocytes were incubated with 5 uM MM 4–64 (AAT Bioquest Inc, CA, USA) in HEPES buffer for 30 min, after which they were washed in HEPES buffer for 10 min. The Olympus FV10i confocal microscope (Olympus, Japan) was used to acquire images (excitation wavelength 558 nm and emission wavelength 734 nm). Images were processed using an Olympus FluoView FV10 (Version 4.2). TTpower was analyzed by imageJ as previously described^{51 (link)}.
For immunofluorescence microscopy, cardiomyocytes were fixed at room temperature for 10 min in 4% polyformaldehyde, permeabilized in 0.2% Triton X-100 for 10 min and blocked with blocking buffer (1% BSA, 22.5 mg/ml glycine, 1 × PBS, 0.1% Tween 20). Cardiomyocytes were then incubated with primary antibodies against JPH2 (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and RYR2 (1:200) (Proteintech Group, China) overnight at 4 °C, and the next day, with Alexa Fluor-labeled secondary antibodies at room temperature for 1 h. Finally, cardiomyocytes were mounted using fluorescent mounting medium and images acquired using the Olympus FV10i confocal microscope. TTpower JPH2 and Pearson correlation coefficient (JPH2 and RYR2) were analyzed by imageJ, as previously described^{51 (link)}.