For early apoptosis detection, a JC-1 fluorescence probe (Beyotime, Jiangsu, China) was used to analyse the mitochondrial membrane potential (ΔΨm) via fluorescence microscopic imaging. After 24 h of treatment, HepG2 cells were stained with JC-1 at 37 °C for 20 min in the dark and then washed twice with JC-1 staining buffer before detection. The average fluorescence intensity was determined by ImageJ software (National Institutes of Health, Bethesda, MD, USA). A decrease in the fluorescence intensity ratio of the JC-1 aggregate (red) to the monomer (green) indicated the loss of ΔΨm.
Jc 1 fluorescence probe
Manufactured by Beyotime
Sourced in China
The JC-1 fluorescence probe is a fluorescent dye used to measure mitochondrial membrane potential in cells. It exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (529 nm) to red (590 nm).
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Lab products found in correlation
Human tnf α elisa kit, by Beyotime (1 mentions)
Cytomics fc500, by Beckman Coulter (1 mentions)
Geneticin g418, by Thermo Fisher Scientific (1 mentions)
Radio immunoprecipitation assay ripa buffer, by Beyotime (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Alginate, by Merck Group (1 mentions)
Na2seo3, by Merck Group (1 mentions)
Cell counting kit 8, by Beyotime (1 mentions)
Dcfh da, by Applygen (1 mentions)
Dcfh da, by Beyotime (1 mentions)
7 protocols using jc 1 fluorescence probe
1
Mitochondrial Membrane Potential Evaluation
2
Mitochondrial Membrane Potential Assay
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Mitochondria membrane potential assay was conducted using JC-1 fluorescence probe (Beyotime, Shanghai, China). TNFα was detected using Human TNFα ELISA Kit (Beyotime).
3
Mitochondrial Membrane Potential Assay
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Cells were treated with 20 and 40 μM BA or the vehicle control for 24 h, followed by incubation with JC-1 fluorescence probe (Beyotime Biotechnology, Shanghai, China) at 37 °C in a humidified incubator with 5% CO2 for 20 min. Then, cells were washed twice with staining buffer, and the pellet was suspended and analyzed using CytomicsTM FC 500 (Beckman Coulter, CA, USA). The emission of JC-1 monomers was detected at 530 nm, and JC-1 aggregates were detected at 590 nm. A loss of aggregates and an increase in monomers signal the disruption of the mitochondrial transmembrane potential (ΔΨm).
4
Quantifying Mycelial Oxidative Stress
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The mycelium ROS (total ROS) level was measured using a Fungus ROS High-Quality Assay kit (GenMed Scientifics, China) according to the manufacturer's instruction with an excitation wavelength of 490 nm and an absorption wavelength of 530 nm. Mitochondrial ROS (mtROS) level was measured using a Mitochondrial ROS Assay kit (GenMed Scientifics, China). Three biological repeats were performed for each sample.
The mitochondrial respiratory chain complex V (F0F1-ATP synthase) activity and specific activity assays were measured using Spectroscopy Quantitative Detection kit (GenMed Scientifics) following the manufacturer's instruction with purified mitochondrial preparations. Meanwhile, the mitochondrial membrane potential (MMP) was measured by a JC-1 fluorescence probe (Beyotime, Shanghai, China) (Wang et al., 2020 (link)).
The mitochondrial respiratory chain complex V (F0F1-ATP synthase) activity and specific activity assays were measured using Spectroscopy Quantitative Detection kit (GenMed Scientifics) following the manufacturer's instruction with purified mitochondrial preparations. Meanwhile, the mitochondrial membrane potential (MMP) was measured by a JC-1 fluorescence probe (Beyotime, Shanghai, China) (Wang et al., 2020 (link)).
5
Mitochondrial Membrane Potential Measurement
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The decrease in mitochondrial
membrane potential is one of the
tokens of apoptosis, which could be detected by the JC-1 fluorescence
probe (Beyotime, Shanghai, China). Hela cells were seeded in a 6-well
plate with selenadiazole diluted by a complete culture medium for
24 h. First, the medium was removed, and PBS was utilized for washing.
Then, we added 2000 μL of JC-1 staining reagent into each well
and put them into a 37 °C, 5% CO2 incubator. Twenty
minutes later, the JC-1 staining reagent was obsoleted, and we added
JC-1 buffer into each well to clear away the extra JC-1 staining reagent.
Ultimately, we put a complete culture medium into each well and observed
the distribution of JC-1 monomers and aggregates using a fluorescence
microscope. For the quantitative experiment, we first digested cells
into a suspension and then repeated the steps mentioned above. Flow
cytometry was applied for detecting fluorescence and a quantitative
analysis.
membrane potential is one of the
tokens of apoptosis, which could be detected by the JC-1 fluorescence
probe (Beyotime, Shanghai, China). Hela cells were seeded in a 6-well
plate with selenadiazole diluted by a complete culture medium for
24 h. First, the medium was removed, and PBS was utilized for washing.
Then, we added 2000 μL of JC-1 staining reagent into each well
and put them into a 37 °C, 5% CO2 incubator. Twenty
minutes later, the JC-1 staining reagent was obsoleted, and we added
JC-1 buffer into each well to clear away the extra JC-1 staining reagent.
Ultimately, we put a complete culture medium into each well and observed
the distribution of JC-1 monomers and aggregates using a fluorescence
microscope. For the quantitative experiment, we first digested cells
into a suspension and then repeated the steps mentioned above. Flow
cytometry was applied for detecting fluorescence and a quantitative
analysis.
6
Evaluating Bioactive Molecule Effects
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Alginate, SO3-Py, and Na2SeO3 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Foetal bovine serum (FBS), high-glucose DMEM, Opti-MEM, penicillin, streptomycin, and geneticin (G418) were provided by Gibco (Grand Island, NY, USA). Cell Counting Kit (CCK)-8, radioimmunoprecipitation assay (RIPA) buffer, and JC-1 fluorescence probe were provided by Beyotime Institute of Biotechnology (Jiangsu, China).
7
Detecting Oxidative Stress and Apoptosis
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After the treatment with different groups combined with US (1 MHz, 3.5 W/cm2), HepG2 cells and Huh7 cells were incubated with DCFH–DA (Applygen Technologies Inc., Beijing, PR China) for 20 min, the concentration of DCFH–DA was 10 mmol/L.
The mitochondrial membrane potential for early apoptosis was detected with a JC-1 fluorescence probe (Beyotime, Jiangsu, China). First, JC-1 liquid was added in HepG2 and Huh7 cells for 20 min without light, and then, JC-1 staining buffer was used to wash the cells twice before taking photos with the fluorescence microscope. ImageJ software (National Institutes of Health, Bethesda, MD, United States) calculated the average intensity of fluorescence.
The mitochondrial membrane potential for early apoptosis was detected with a JC-1 fluorescence probe (Beyotime, Jiangsu, China). First, JC-1 liquid was added in HepG2 and Huh7 cells for 20 min without light, and then, JC-1 staining buffer was used to wash the cells twice before taking photos with the fluorescence microscope. ImageJ software (National Institutes of Health, Bethesda, MD, United States) calculated the average intensity of fluorescence.
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