The JC-1 method was used to assess the mitochondrial membrane potential (MMP) of spermatozoa. The sperm concentration was adjusted to 1×106/mL with Biggers, Whitten, and Whittingham (BWW) medium (95 mM NaCl, 4.6 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.6 mM glucose, 0.27 mM sodium pyruvate, 20 mM acid-free HEPES, 25 mM NaHCO3, 44 mM lactic acid, and 0.3% BSA [pH 7.4]), and the sperm suspension was added to the MMP assay kit (Solarbio, China) according to the following instructions. First, 0.5 mL of washed sperm was added to the MMP assay kit (Solarbio, China). The sperm concentration was adjusted to 1×106/mL with BWW medium, and the MMP assay kit (Solarbio, China) was used according to the manufacturer’s instructions. Then, 0.5 mL of JC-1 working solution was added to 0.5 mL of washed sperm suspension and incubated at 37 °C for 20 min. After incubation, spermatozoa were washed and resuspended with JC-1 staining buffer. The percentage of red or orange sperm/total sperm was calculated using flow cytomete (Celula Medical Technology Co, China) and recorded as MMP.
Mmp assay kit
Manufactured by Solarbio
Sourced in China
The MMP assay kit is a tool used to measure the activity of matrix metalloproteinases (MMPs), a group of enzymes involved in the degradation of the extracellular matrix. The kit provides the necessary reagents and protocols to quantify the enzymatic activity of specific MMPs in a sample.
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Lab products found in correlation
Confocal laser scanning microscope, by Olympus (2 mentions)
Facsaria sorp, by BD (1 mentions)
Atp assay kit, by Beyotime (1 mentions)
Mda assay kit, by Solarbio (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Confocal microscope system and camera, by Nikon (1 mentions)
Facsdiva software, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Dna quantification kit, by Solarbio (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
5 fluorouracil 5 fu, by MedChemExpress (1 mentions)
Facscalibur, by BD (1 mentions)
8 protocols using mmp assay kit
1
Mitochondrial Membrane Potential in Sperm
2
Mitochondrial Membrane Potential Assay
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MMP was measured by a fluorescent probe JC‐1 using the MMP assay kit (Solarbio) based on the manufacturer’s directions. In brief, the isolated mitochondria were added into JC‐1 staining working solution. After the contents were mixed, the fluorescence intensity of both mitochondrial JC‐1 monomers (green fluorescence; excitation wavelength (λex) 490 nm, emission wavelength (λem) 530 nm) and aggregates (red fluorescence; λex 525 nm, λem 590 nm) were measured by a Multi‐Mode Microplate Reader (Synergy H1). The MMP was calculated according to the fluorescence ratio of red to green per milligram of protein.
3
Mitochondrial Membrane Potential Assay
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The ΔΨm of RECs was detected using an MMP assay kit (CA1310, Solarbio, Beijing, China) following the manufacturer’s instructions. Briefly, cells were collected using 2.5% trypsin solution and incubated at 37 °C for 20 min with 500 μL 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1). The cells were then measured for ΔΨ m by flow cytometry (FACSAria SORP; BD Bioscience) and fluorescence microscopy (DMi8; Leica). FlowJo v10.7.1 (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze ΔΨm in cells.
4
Comprehensive Biochemical Analyses of Samples
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All tests were conducted per the respective manufacturers’ instructions for each kit and were obtained from the following vendors: reactive oxygen species (ROS) assay kit (CA1410, Solarbio, Beijing, China), malondialdehyde (MDA) assay kit (BC0025, Solarbio, Beijing, China), pyruvate (PA) content assay kit (BC2205, Solarbio, Beijing, China), lactic acid (LA) assay kit (A019-2-1, Nanjing Jiancheng, Nanjing, China), ATP assay kit (S0026, Beyotime, Shanghai, China), MMP assay kit (M8650, Solarbio, Beijing, China), and bicinchoninic acid (BCA) protein assay kit (PA115, TAINGEN, Beijing, China). Finally, the related indexes were detected by a multifunctional enzyme label instrument (Synergy H1, American Berten, VT, USA).
5
Mitochondrial Function Evaluation Protocol
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The MMP was measured using an MMP assay kit (Solarbio, Beijing, China). Cells were stained with the unique fluorescence probe JC-1 at 37 °C for 20 min, and then were washed twice with phosphate-buffered saline. The fluorescence intensity of the cells was observed using a flow cytometer (BD FACSCalibur, NJ, USA) and a confocal laser scanning microscope (Olympus, Japan). Then, the average fluorescence intensity of green monomers and red aggregates was determined, and the ratio was calculated.
MitoTracker™ was used to detect changes in mitochondrial dynamics. For mitochondrial labeling, the cell samples were incubated with a 100 nM solution of MitoTracker™ Green FM (Thermo Fisher Scientific, MA, USA) for 30 min at 37 °C, 5% CO2. Images were obtained using a Nikon confocal microscope system and camera (Nikon Instruments, NY, USA). The fluorescent dye and length of mitochondria were measured using Image J software. We selected 10 random fluorescence fields from each group.
To track mPTP opening, we treated HCAECs with tetramethylrhodamine ethyl ester, based on our previous study. The fluorescent signal of tetramethylrhodamine ethyl ester was determined using a Nikon confocal microscope system and camera.
MitoTracker™ was used to detect changes in mitochondrial dynamics. For mitochondrial labeling, the cell samples were incubated with a 100 nM solution of MitoTracker™ Green FM (Thermo Fisher Scientific, MA, USA) for 30 min at 37 °C, 5% CO2. Images were obtained using a Nikon confocal microscope system and camera (Nikon Instruments, NY, USA). The fluorescent dye and length of mitochondria were measured using Image J software. We selected 10 random fluorescence fields from each group.
To track mPTP opening, we treated HCAECs with tetramethylrhodamine ethyl ester, based on our previous study. The fluorescent signal of tetramethylrhodamine ethyl ester was determined using a Nikon confocal microscope system and camera.
6
Evaluating Luteolin's Impact on Mitochondrial Membrane Potential
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MMP changes were detected in the cells using an MMP assay kit (M8650, Solarbio, Beijing, China) and flow cytometry (19 (link)). The cells were treated with 10, 40, and 70 µM luteolin for 24 h or 48 h. Then, the treated cells were resuspended in a complete media and stained with a JC-1 fluorescence working solution. MMP changes were measured with the FACSCanto II flow cytometer (Becton Dickinson and Company, Franklin Lakes, United States), and the fluorescence intensity was analyzed using the FACSDiva software (version 6.1.3; Becton Dickinson and Company, Franklin Lakes, United States). The relative ratio of red fluorescence to green fluorescence was applied for quantitative analysis of the MMP changes. The data were normalized as fold changes in comparison to the DMSO group. Experiments were repeated in HGC-27 cells (n=12), MFC cells (n=11), and MKN-45 cells (n=4).
7
Evaluating JAC and 5-FU Cytotoxicity
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Cell-level dimethyl sulfoxide (DMSO) (Solarbio, Beijing, China) was used to dissolve JAC (HerbPurify, Chengdu, China) and 5-fluorouracil (5-FU) (Med Chem Express, Princeton, NJ, USA) and the solutions were stored at −20°C. Cell Counting kit-8 (CCK-8), Annexin V-FITC/PI apoptosis kit, MMP assay kit, and DNA quantification kit were acquired from Solarbio. ROS assay kit was obtained from Beyotime (Shanghai, China).
8
Mitochondrial Membrane Potential Assay
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The mitochondrial membrane potential was measured using an MMP assay kit (Solarbio, Beijing, China). Cells were stained with the unique fluorescent probe JC-10 at 37 °C for 20 min, and then were washed twice with phosphate-buffered saline [63 ]. The fluorescence intensity of the cells was observed using a flow cytometer (BD FACSCalibur, NJ, USA) and a confocal laser scanning microscope (Olympus, Japan). Then, the average fluorescence intensity of green monomers and red aggregates was determined, and the ratio was calculated. To track mPTP opening, we treated cardiomyocytes with tetramethylrhodamine ethyl ester, based on our previous research [12 (link),64 ]. The fluorescent signal of tetramethylrhodamine ethyl ester was determined using a Nikon confocal microscope system and camera.
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