Jc 1 staining assay kit

The JC‐1 staining assay kit (Beyotime) was used to measure mitochondrial membrane potential (∆Ψm). JC‐1 is a fluorescent probe widely used for detecting ∆Ψm. It emits red fluorescence at high ∆Ψm and green fluorescence at low ∆Ψm. In brief, primary cardiac cells were incubated with JC‐1 (37°C, 20 min), washed three times, and immediately observed and imaged using laser scanning confocal microscopy at emission wavelengths of 530 and 590 nm.

The alteration of the ΔΨm in BMMSCs was analyzed using a JC-1 staining assay kit according to the manufacturer's instructions (Beyotime). Briefly, BMMSCs were collected, rinsed with PBS and stained with JC-1 (20 μg/ml) for 30 min at 37 °C in the dark. Cells were rinsed with staining buffer twice and subjected to FACS.

Cells were cultured overnight after plating at a concentration of 3 × 10⁵ cells/well. Then, the cells were treated with 1% DMSO, 5 μg/mL LPS, 5 μg/mL LPS plus shNC, or 5 μg/mL LPS plus shCDK6-AS1 for 24 h. Cells were collected by centrifugation and washed twice with pre-chilled phosphate buffer saline. The cells were stained using the JC-1 staining assay kit (Beyotime, catalog number: C2006) or the annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Vazyme, catalog number: A211-01) according to the manufacturer’s instructions. Subsequently, mitochondrial membrane potential and apoptosis were detected via flow cytometry. Three independent experiments were performed.

The mitochondrial membrane potential was assessed using a JC-1 staining assay kit (Beyotime, China, C2005). The fluorescent dye JC-1 accumulates in the mitochondrial matrix in two forms depending on mitochondrial membrane potential: JC-1 aggregates (red) in healthy mitochondria and JC-1 monomers (green) in depolarized mitochondria. The frozen sections were pretreated with PBS for 10 min and then stained with a JC-1 probe (10 µg/mL) at 37 °C for 30 min in the dark cassette [31 ]. After washings with PBS three times, the sections were observed using a fluorescence microscope (Leika, Germany). Five random images were captured per sample to examine the fluorescence intensity of the J-monomers and the J-aggregates.

A JC-1 staining assay kit (C2006, Beyotime) was used to detect alterations in the mitochondrial membrane potential. Cells were resuspended in 500 μL RPMI-1640 medium and then mixed with 500 μL JC-1 working solution. After incubation for 20 min at 37°C while protect from light, the cells were washed twice with JC-1 staining buffer, and resuspended in 300 μL JC-1 staining buffer, and then analyzed by a NovoCyte FACS (Cat: 1300, ACEA, San Diego, CA, USA).

MMP was measured by using the JC-1 staining assay kit (Cat# C2006, Beyotime, China) according to the manufacturer's instructions. Briefly, the cells were incubated with JC-1 fluorescent probe for 20 minutes in the dark at 37°C. After washing with PBS, cell images were detected using a fluorescent microscope (IX83, Olympus Co., Japan). The average fluorescence intensity (AFI) was quantified by using ImageJ.

A JC-1 staining assay kit (C2006, Beyotime) was used to detect mitochondrial membrane potential. 1 × 10⁶ cells were resuspended in 200 µL of medium and then mixed with 200 µL of the JC-1 working solution. After incubation for 20 min at 37 °C in the dark, cells were washed twice with the JC-1 staining buffer, resuspended in 300 µL of the JC-1 staining buffer, and analyzed using a flow cytometer NovoCyte FACS (Cat#: 1300, ACEA, San Diego, CA, USA). Green (JC-1 monomer) and red (JC-1 aggregates) fluorescence were detected within the FITC-channel (Ex: 488 nm/Em: 519 nm) and PE-channel (Ex: 488 nm/Em: 578 nm), respectively. The mean fluorescence intensity (MFI) was measured, and then the MFI ratio of green/red was counted.