Macrophages were washed with PBS and loaded with probe JC-1 (Sigma). After incubation for 20 min, cells were washed with serum-free media and collected in FACS tubes. The cells were centrifuged at 1000 × g for 5 min under 4°C and re-suspended in ice-cold PBS. This procedure was repeated once and green fluorescent intensity (JC-1 monomer) was determined by fluorescent microplate reader (Molecular Devices M3) with Ex 490 nm/Em 530 nm and red fluorescent intensity (JC-1 aggregates) with Ex 525 nm/Em 590 nm. Mitochondrial membrane potential was calculated as red fluorescence intensity divided by green fluorescent intensity and was normalized to protein concentrations of cellular lysates.
Jc 1 probe
Manufactured by Merck Group
Sourced in United States, Germany
The JC-1 probe is a fluorescent dye used to measure the mitochondrial membrane potential in cells. It can be used to assess cell health and function.
Automatically generated - may contain errors
Lab products found in correlation
N acetylcysteine nac, by Merck Group (2 mentions)
Mouse cd4 t cell isolation kit, by Miltenyi Biotec (2 mentions)
Facscalibur, by BD (2 mentions)
Ve cadherin, by Santa Cruz Biotechnology (1 mentions)
Propofol, by Merck Group (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Evans blue dye, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
X71 u rfl t fluorescence microscope, by Olympus (1 mentions)
α cd4 pe, by Thermo Fisher Scientific (1 mentions)
Calibrite beads, by BD (1 mentions)
Canto 2, by BD (1 mentions)
Annexin 5 apc, by BD (1 mentions)
Caspase 3 colorimetric assay kit, by R&D Systems (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Fluorescein diacetate, by Merck Group (1 mentions)
Nile red, by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
18 protocols using jc 1 probe
1
Mitochondrial Membrane Potential Assay
2
Propofol-induced Mitochondrial Dysfunction
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Human HMGB1 (recombinant) was from R&D Systems (Minneapolis, USA). Sterile 0.1% BSA/PBS was used to resuspend the protein. Propofol, dextran conjugated with fluorescein isothiocyanate (FITC)(10-kDa), fluorescent probe JC-1, and Evans blue dye were procured from Sigma-Aldrich (St. Louis, MO). MitoSOX was supplied by Molecular Probes (Eugene, OR). VE-Cadherin and GAPDH primary antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, CA). ZO-1 primary antibodies were obtained from ProteinTech Group (Chicago, IL, USA).
3
Measuring Mitochondrial Membrane Potential
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JC-1 probe (Sigma-Aldrich; Merck KGaA) was used to investigate the mitochondrial membrane potential following erastin treatment. HGC-27 cells were incubated with JC-1 (20 µg/ml) at 37°C for 20 min and then washed twice with PBS. Treated cells were observed by an X71 (U-RFL-T) fluorescence microscope (Olympus Corporation, magnification, ×40).
4
Mitochondrial Membrane Potential Assay for Candida
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JC‐1 probe (Sigma) was used to ascertain changes in mitochondrial membrane potential (∆Ψm). C. albicans (105 cfu/mL) cocultured with honey isolate were stained with 1 mg/ml of JC‐1 at 37°C for 20 min (Kosgey et al., 2020 ; Ma et al., 2016 ; Pina‐Vaz et al., 2001 ). Sodium azide (1mM) (Tianjin Fuchen), a fungal respiratory inhibitor, was used as positive control (Pina‐Vaz et al., 2001 ). Immediately after staining, the mean of the fluorescence intensities was captured using an laser scanning confocal microscope (Olympus FluoViewFV500/IX) and quantitative analysis of mitochondrial membrane potential (ratio of green/red fluorescence) from three independent experiments using ImageJ software, and the ratio of aggregated JC‐1 (FL2) to monomer of JC‐1 (FL1) intensity was calculated.
5
Multiparametric Flow Cytometry Analysis
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Quantification of cell number and assessment of cell death were achieved by addition of both a defined number of fluorescently labeled CaliBRITE beads (BD) and propidium iodide (PI; 1:3,000; Sigma-Aldrich) to cell suspensions. For assessment of apoptosis, cells were co-stained with APC Annexin V (BD) according to manufacturer’s instructions before addition of beads and PI. Cells obtained from LNs of Shigella-infected mice were co-stained with α-CD19 FITC (1:100; eBioscience) and α-CD4 PE (1:100; eBioscience). Loss of MMP was assessed by use of the JC-1 probe (mitochondrial staining kit; Sigma-Aldrich) according to manufacturer’s instructions. Samples were acquired on a FACSCalibur or Canto II (BD) and analyzed with FlowJo software.
6
Dihydroartemisinin-Induced Apoptosis Mechanisms
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DHA (Cat: A2679) was purchased from LKT-Laboratory. Inc., Minnesota, USA. MTT (Cat: M5655), dimethyl sulfoxide (DMSO) (Cat: PHR1309), JC-1 probe (Cat: T4069), N-acetylcysteine (NAC) (Cat: 1009005) were from Sigma-Aldrich Munich, Germany. Annexin V-FITC/PI apoptosis detection kit (Cat: 4830-01-K), Caspase-3 Colorimetric Assay kit (Cat: BF3100) were purchased from R & D System. Marker Gene™ Live Cell Fluorescent ROS Detection Kit (Cat: M1049), Antibodies against Bcl2 (sc-7382), Bax (sc-13156), cytochrome c (Cat: Sc-13561), and also horseradish peroxidase secondary antibodies (sc-358923) were purchased from Santa Cruz Biotechnology Co.
7
Mitochondrial Membrane Potential in CD4+ T Cells
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CD4+ T cells were sorted from the spleen of naïve C57BL/6 mice using the Mouse CD4+ T cell Isolation Kit (Miltenyi, Bergisch Gladbach, Germany), seeded in 12-well plates, and treated with 5 μg/mL ConA alone or 5 μg/mL ConA + ATO (1 and 2 μM) for 24 h. JC-1 staining was performed to monitor the change of mitochondrial membrane potential. Briefly, the CD4+ T cells were incubated with 1640 RPMI medium containing 10 mg/mL JC-1 probe (Sigma) for 30 min at 37 °C. After washing three times with PBS, the stained cells were examined by flow cytometry with Beckman Cytoflex S.
8
Lipid-Polymer Nanoparticle Synthesis and Characterization
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Poly(d,l-lactic-co-glycolic acid) (PLGA) 50:50 DL (24–38kDa), polyvinyl alcohol (PVA) (MW 9000–10,000, 80% hydrolyzed), ethyl acetate (Sigma Aldrich), acetone, coumarin-6, JC-1 probe, fluorescein diacetate and propidium iodide were purchased from Sigma-Aldrich; Nile Red (TCI chemicals); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol (Chol) and 1.2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000) were purchased from Avanti PolarLipids; Selumetinib was kindly offered by AstraZeneca; sorafenib was kindly provided by Bayer HealthCare Pharmaceutical; and triantennary N‐acetylgalactosamine (GalNAc) cluster was kindly offered by Ionis Pharmaceuticals.
9
Cannabinoid-Induced Mitochondrial Apoptosis
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The study protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University. Procedures involving animals and their care were conducted in conformity with NIH guidelines (NIH Pub. No. 85-23, revised 1996) and was approved by Animal Care and Use Committee of Renmin Hospital of Wuhan University. Experimental animals: 30 healthy SPF male C57BL/6 mouse, weighing 20–25 g, were purchased from Beijing vitonghua laboratory animal technology co., Ltd. Growth temperature was 23.0±2.0°C, environment humidity: 45–50%, illumination time: 12 hrs a day; free food, water, adaptive feed for a week was provided.
Primary reagents: Endogenous cannabinoids 2-AG injection was purchased from Jiangsu Rui Heng pharmaceutical co., Ltd. 2-AG was purchased from Shanghai Wei Huan Biotech Co., Ltd. AV/PI cell apoptosis dual infection kit was purchased from BD company. The JC-1 probe was purchased from Sigma in the United States. BCA protein concentration determination kit and ECL luminescence kit were purchased from Biyuntian Institute of Biotechnology. Mitochondrial protein extraction kit was purchased from Beijing Kangwei Biotechnology co., Ltd. AIF antibody, Endo G antibody, BNIP3 antibody and trader-actin antibody were all purchased from ABcom company; others are domestic analytical pure reagents.
Primary reagents: Endogenous cannabinoids 2-AG injection was purchased from Jiangsu Rui Heng pharmaceutical co., Ltd. 2-AG was purchased from Shanghai Wei Huan Biotech Co., Ltd. AV/PI cell apoptosis dual infection kit was purchased from BD company. The JC-1 probe was purchased from Sigma in the United States. BCA protein concentration determination kit and ECL luminescence kit were purchased from Biyuntian Institute of Biotechnology. Mitochondrial protein extraction kit was purchased from Beijing Kangwei Biotechnology co., Ltd. AIF antibody, Endo G antibody, BNIP3 antibody and trader-actin antibody were all purchased from ABcom company; others are domestic analytical pure reagents.
10
Apoptosis and Mitochondrial Assays
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For the PI and ANXA5 staining assay, cells were harvested after drug treatment or transfection as indicated and washed with PBS. Then cells were placed in tubes and stained with FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, 556547) according to the manufacturer´s protocol.
For the mitochondrial membrane potential assay, a JC-1 probe (5 μM; Sigma-Aldrich, T4069) was used to measure mitochondrial depolarization in CCC-HEH-2 cells. After treatment, cells were digested with trypsin and incubated with an equal volume of JC-1 solution (5 μg/mL) at 37°C for 20 min in the dark. After washing, resuspend cells with 1× buffer.
A FACSCalibur cytometer (BD Biosciences, USA) was employed for analysis.
For the mitochondrial membrane potential assay, a JC-1 probe (5 μM; Sigma-Aldrich, T4069) was used to measure mitochondrial depolarization in CCC-HEH-2 cells. After treatment, cells were digested with trypsin and incubated with an equal volume of JC-1 solution (5 μg/mL) at 37°C for 20 min in the dark. After washing, resuspend cells with 1× buffer.
A FACSCalibur cytometer (BD Biosciences, USA) was employed for analysis.
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