The method was as described in our previous study [19 (link)]. ROS levels in RAECs were measured by a DCFH-DA fluorescent probe according to the ROS assay kit protocol (Beyotime, China). TUNEL staining of paraffin sections was performed according to the TUNEL kit protocol (Beyotime, China). JC-1 staining of RAECs was performed according to the JC-1 assay kit protocol (Solarbio, China).
Jc 1 assay kit
Manufactured by Solarbio
Sourced in China
The JC-1 assay kit is a fluorescence-based tool used to measure mitochondrial membrane potential in cells. It provides a reliable method for evaluating mitochondrial function and health.
Automatically generated - may contain errors
11 protocols using jc 1 assay kit
1
ROS, TUNEL, and JC-1 Assays in RAECs
2
Mitochondrial Membrane Potential Assessment
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Mitochondrial membrane potential was determined using JC-1 assay kit (Solarbio, China). After 24 h of LPS (1 μg/mL) exposure, the cell culture was discarded and was replaced with JC-1 staining solution (100 ml/well). Then, they were incubated at 37°C for 30 min in darkness and washed with PBS, JC-1 fluorescence was detected and photographed using a LSM-800 with Airyscan. For red fluorescent of J-aggregates, the wavelength of excitation/emission were set as 525 and 590 nm, respectively, and for the fluoresce green of J-monomers, the excitation/emission were 490/530 nm, respectively.
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3
Mitochondrial Membrane Potential Assay
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JC-1 is a fluorescent probe frequently used for detecting the MMP. Experiments were performed with the JC-1 assay kit (Cat# M8650; Solarbio). Huc-MSCs were treated following exposure to PBS, RSL3 (6 μM), or RSL3 (6 μM) + Tz-A6 peptide (1 μM) + ROS Nano@Feb-1 (Feb-1 concentration of 0.1 nM). After treatment, plates were washed twice with PBS. Subsequently, cells were incubated in a staining solution for 30 min and placed in the 37 °C incubator. After staining, cells were washed twice. The staining buffer was replaced with MEM medium (Cat#C11095500BT; Gibco-Invitrogen) and plates were placed under a fluorescence microscope (Leica, DMi8, GER). Finally, images were acquired using fluorescence microscopy and quantified using Image J (NIH).
4
Mitochondrial Potential Evaluation in AGS Cells
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AGS cells were seeded in 3.5 cm Petri dishes (1 × 105 cells/dish) and cultured overnight. For 0, 3, 6, 12, and 24 h, the cells were exposed to 36.54 µM ISO. The JC–1 Assay Kit was used to assess the mitochondrial membrane potential of AGS cells (Solarbio). After centrifugation, AGS cells were collected and incubated with 1 mL JC–1 at 37 °C for 30 min. Then, AGS cells were suspended in a 1 mL binding solution two times and were detected by flow cytometry.
5
Measuring Cellular Oxidative Stress and Apoptosis
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The level of ROS in LO2 cells was measured by DCFH-DA fluorescent probe according to the ROS Assay Kit protocol (Beyotime, China). TUNEL staining for the liver paraffin sections was performed according to the TUNEL Kit protocol (Roche, USA). JC-1 staining for LO2 cells was performed according to the JC-1 assay Kit protocol (Solarbio, China).
6
Mitochondrial Function Evaluation in Macrophages
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After 24 h of treatment with ACAP, H2O2 (0.6 mM) was used to stimulate RAW 264.7 cells for 24 h; the cell culture was replaced with JC-1 staining solution. After incubating at 37°C for 30 min in darkness and washed with PBS, the fluorescence was photographed using an LSM-800 with Airyscan using the JC-1 assay kit (Solarbio, China). For red fluorescent of J-aggregates and the fluoresce green of J-monomers, the wavelength of excitation/emission was set as 525/590 nm and 490/530 nm, respectively (20 (link)).
7
Mitochondrial Membrane Potential Assessment
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We
examined the mitochondrial membrane potential (MMP) of hGCs using
JC-1 staining and flow cytometry with a JC-1 Assay Kit (M8650, Solarbio)
as described in the product manual. Briefly, 1 mL of cell culture
medium and 1 mL of JC-1 staining solution was added to the cells cultured
for 2 days in the 2D or 3D system (the method of cell collection in
the 3D culture system was the same asSection 2.9 ), incubated for 20 min at 37 °C,
washed twice with buffer, and observed by fluorescence microscopy
(IX73, Olympus) or analyzed by flow cytometry. At high MMP, JC-1 aggregated
in the mitochondrial matrix and formed polymers (JC-1 aggregates),
which can produce red fluorescence. At low MMP, JC-1 cannot aggregate
in the mitochondrial matrix, when JC-1 was a monomer, which can produce
green fluorescence. The relative MMP ratio was calculated as red fluorescence
intensity/green fluorescence intensity.
examined the mitochondrial membrane potential (MMP) of hGCs using
JC-1 staining and flow cytometry with a JC-1 Assay Kit (M8650, Solarbio)
as described in the product manual. Briefly, 1 mL of cell culture
medium and 1 mL of JC-1 staining solution was added to the cells cultured
for 2 days in the 2D or 3D system (the method of cell collection in
the 3D culture system was the same as
washed twice with buffer, and observed by fluorescence microscopy
(IX73, Olympus) or analyzed by flow cytometry. At high MMP, JC-1 aggregated
in the mitochondrial matrix and formed polymers (JC-1 aggregates),
which can produce red fluorescence. At low MMP, JC-1 cannot aggregate
in the mitochondrial matrix, when JC-1 was a monomer, which can produce
green fluorescence. The relative MMP ratio was calculated as red fluorescence
intensity/green fluorescence intensity.
8
Mitochondrial Membrane Potential Assay
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Check for changes in MMP (mitochondrial membrane potential) with the JC-1 Assay Kit [26 (link)] (Solarbio, No: #M8650) according to the manufacturer's instructions, A total of 5 × 104 cells were seeded in 6-well plates, and LX-2 cells were treated with acacetin at various concentrations for 24 h. Then, after incubation according to the manufacturer's instructions, the samples were analyzed by flow cytometry.
9
Mitochondrial Membrane Potential Assay
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MMP was detected using JC-1 assay kit (Solarbio) following its guidance. Simply, HepG2
and AML12 cells were incubated with JC-1 probes at 37°C for 20 min. Accumulation of JC-1
was determined using flow cytometry analysis (Becton Dickinson).
and AML12 cells were incubated with JC-1 probes at 37°C for 20 min. Accumulation of JC-1
was determined using flow cytometry analysis (Becton Dickinson).
10
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential was determined using the JC-1 assay kit (Solarbio, CN). The decrease of mitochondrial membrane potential is a landmark event in the early stage of apoptosis. The JC-1 dye aggregates in the mitochondria of healthy cells and emits red fluorescence. However, in unhealthy cells, due to the drop or loss of mitochondrial membrane potential, the JC-1 dye cannot aggregate in the mitochondria, and remains as monomers in the cytoplasm and emits green fluorescence. The transition of fluorescence is usually used as an indicator of early apoptosis. 72 h after transfection, cells were washed with PBS, stained with JC-1 for 20 min, observed under the fluorescence microscope (Nikon, Japan).
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