Phalloidin fluorescein isothiocyanate phalloidin fitc

Neonatal rat cardiomyocytes were seeded in a 96-well collagen I plate at a density of 5000 cells/well. They were incubated overnight and then transferred to a serum-free medium for one day. Next, the cells were treated with the indicated concentrations of compounds and 10 μM isoproterenol, and then incubated for 2 days. To quantify cardiomyocyte hypertrophy via filamentous actin staining, NRCMs were fixed with 4% paraformaldehyde (Wako) and permeabilized with 0.1% Triton X-100 (MP Biomedicals, France), followed by blocking with 1% bovine serum albumin (Wako) and staining with 2 μM phalloidin–fluorescein isothiocyanate (phalloidin-FITC; Sigma-Aldrich, Japan) and 10 μM Hoechst 33258 (Dojindo). Quantification of FITC-fluorescence intensity and cell number was performed using an IN Cell Analyzer 6000 (GE Healthcare Japan). The signals from obtained images were quantitated by means of a customized image analysis algorithm using the IN Cell Developer Toolbox (GE Healthcare Japan). Hypertrophic scores were calculated as the product of FITC-fluorescence intensity and cell area per cell as programmed by GE Healthcare technical support experts.

Hyaluronic acid (MW 100,000 Da and 200,000 Da) was purchased from Yuanye Biotechnology Co., Ltd., (Shanghai, China). Cystamine dihydrochloride was obtained from Macklin Biotechnology Co., Ltd., (Shanghai, China). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), isoamyl acetate, and sodium pentobarbital were obtained from Aladdin Industrial Co., Ltd., (Shanghai, China). Alamar Blue was bought from Maokang Biotechnology Co., Ltd., (Shanghai, China). Glutaric dialdehyde solution, fluorescein isothiocyanate-phalloidin (FITC-phalloidin), and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).