The JC-1 chemical probe was used to visualize ΔΨm. A commercial JC-1 kit (Cayman Chemical, Ann Arbor, MI) was used that comprises a monomer that yields green fluorescence at low ΔΨm (≤−100 mV) and a polymer that yields red fluorescence at high ΔΨm (≥−140 mV). The frozen JC-1 reagent was equilibrated to room temperature. The dispensed reagent was mixed with 225 µL D-MEM as a stock solution and stored at −20 °C prior to use. After thawing 25 µL of the stock solution, it was mixed with 500 µL D-MEM as a working solution. The D-MEM was removed from the glass culture dish, and the working solution was added to the cells in a cell incubator for 20 min. After incubation, the solution was removed from the dish and the cells were washed twice with PBS. Fresh D-MEM was applied for observations under a fluorescence microscope.
Jc 1 kit
Manufactured by Cayman Chemical
Sourced in United States
The JC-1 kit is a fluorescent probe for detecting changes in mitochondrial membrane potential. It is a cell-permeant cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (529 nm) to red (590 nm). The JC-1 kit is commonly used in research applications to monitor mitochondrial function and health.
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Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Cell culture medium, by Thermo Fisher Scientific (1 mentions)
Cck 8 kit, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Indocyanine green icg, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Sodium oleate, by Merck Group (1 mentions)
Alkaline phosphatase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Bodipy fl c16, by Thermo Fisher Scientific (1 mentions)
Alexa fluor tagged secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Celllight mitochondria rfp, by Thermo Fisher Scientific (1 mentions)
Shrna plasmid, by OriGene (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Bcip nbt, by Merck Group (1 mentions)
8 protocols using jc 1 kit
1
Measurement of Mitochondrial Membrane Potential
2
Sperm Mitochondrial Activity Assay
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Sperm mitochondrial activity was evaluated by 5, 5', 6, 6'-tetrachloro-1-1',3,3'-tetraethyl-benzami-dazolocarbocyanin iodide known as JC-1 kit (Cayman, Item NO:10009172). In this assay, an equal volume of sperm suspension was mixed with JC-1 solution (concentration of 50 times diluted) for 30-40 min at 37 C in dark conditions. Then, the suspension of the cells was centrifuged at 400 g for 5 min and analyzed by a fluorescence microscope (Olympus Co., Tokyo, Japan) at x1000 magnification. In cells with healthy mitochondria, JC-1 formed complexes known as J-aggregates, and the midpiece was seen in red, while in cells with low MMP, JC-1 remained in a monomeric form and was seen in green.
3
Mitochondrial Membrane Potential Assay
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To assess cell health, we utilized a dye that is permeable to the mitochondrial and will fluoresce red or green depending on the membrane potential of the mitochondria. A high mitochondrial membrane potential (ΔψM) is representative of a healthy cell, whereas a low ΔψM is indicative of an unhealthy cell. The higher the ratio, the greater the polarization of the mitochondrial membrane. We utilized the JC-1 kit (Cayman Chemical Co.) for this purpose. The cells were plated at approximately 105 cells/well and allowed to adhere for 24 h. Exposure was initiated by removing the growth media from each well and adding the treatment media with CdCl2 (1 μM), or the vehicle (water). The cells were returned to the incubator for 48 h at 37°C and 5% CO2. Following exposure, the cells were prepared according to manufacturer instructions; staining was initiated by the addition of 10 μ JC-1 dye and the cells were returned to the incubator for 20 min. The fluorescence intensity was measured using the BioTek plate reader at 535ex/595em to measure JC-1 aggregates (healthy cells; green) and then a second reading using 485ex/535em which measures JC-1 monomers (apoptotic/unhealthy cells; red).
4
Multimodal Cell Imaging and Cytotoxicity Assessment
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Ethanol, stannum powder (10 μm, 99% trace metals basis), CCK-8 kit, indocyanine green (ICG), 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), calcein-AM, and propidium iodide (PI) were purchased from Sigma-Aldrich. Phosphate-buffered saline (PBS, pH 7.4, 10 mM), penicillin–streptomycin, trypsin–EDTA, fetal bovine serum (FBS), and cell culture medium were brought from Gibco Life Technologies (AG, Switzerland). JC-1 Kit was purchased from Cayman Chemical. 1,2-Distearoyl-sn-glycero-3-phosphoEthanolamine-N-[methoxy (poly-ethylene glycol)] (DSPE-PEG, MW 5000 Da, PG1-DS-5 k) was obtained from Nanocs, Inc. The other reagents were analytically pure and no need further purification for use. Ultrapure water (18.2 MΩ cm) was applied in all experiments.
5
Intracellular ROS and Mitochondrial Membrane Potential in RBMCs
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The intracellular ROS level was determined using CellROX® oxidative stress reagents (C10422, Thermo Fisher Life Technologies Ltd., Tokyo, Japan). The cells were washed with PBS three times and incubated in medium containing 5 μM CellROX® oxidative stress reagents for 30 min at 37 °C. RBMCs were collected by trypsinization and then transferred into a 96-well plate. ROS levels on RBMCs of the test and control NANOZR disks were stained and observed by confocal laser scanning microscopy (LSM700, Zeiss, Oberkochen, Germany).
Mitochondrial membrane potential (ΔψM) is an important parameter for mitochondrial function in understanding the relationship between ROS and RBMCs. The JC-1 kit (10009172, Cayman, Ann Arbor, MI, USA) was used to determine if UV treatment caused changes in mitochondrial membrane potential of RBMCs. The mitochondrial membrane potential of RBMCs at the test and control NANOZR surface was visualized using a confocal laser scanning microscope (LSM700).
Mitochondrial membrane potential (ΔψM) is an important parameter for mitochondrial function in understanding the relationship between ROS and RBMCs. The JC-1 kit (10009172, Cayman, Ann Arbor, MI, USA) was used to determine if UV treatment caused changes in mitochondrial membrane potential of RBMCs. The mitochondrial membrane potential of RBMCs at the test and control NANOZR surface was visualized using a confocal laser scanning microscope (LSM700).
6
Mitochondrial membrane potential measurement
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MMP staining was performed using a 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolyl-carbocyanine iodide (JC-1) kit (Cayman, MI, USA) according to the manufacturer’s instructions [29] (link), [33] (link). JC-1 is a cationic fluorescent dye that accumulates in mitochondria in a potential dependent manner. When MMP is high, JC-1 accumulates in the mitochondria and forms aggregates demonstrating red fluorescence. When MMP is low, JC-1 exists in the cytoplasm as monomers displaying green fluorescence. Briefly, the cells were incubated with PEP-1-PON1 protein (0.3 µM) for 1 h, and then exposed to H2O2 (2 mM) for 30 min. As above, images were produced using a fluorescence microscope (Nikon eclipse, 80i, Japan).
7
Evaluating Sperm Mitochondrial Potential
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The mitochondrial membrane potential (MMP) was evaluated using a JC1 kit (Cayman, Ann Arbor, MI, USA). First, 25 μL of the sample was mixed with 25 μL of JC-1 dye in a microtube and incubated in a CO2 incubator at 37°C for 15–30 minutes. After incubation, 100 μL of phosphate-buffered saline was added to the samples. Each sample was centrifuged at RT for 5 minutes at 400 ×g and then the supernatant was gently removed. Then, 10 μL of the sample was placed on a slide and a smear was prepared. After the smear was dried, 200 spermatozoa were assessed with a fluorescent microscope under ×1,000 magnification at 520–570 nm. Sperm cells with a red or shiny middle piece were considered to have high mitochondrial potential, or JC-1(+), and sperm cells with a green middle piece were considered to have low mitochondrial potential, or JC-1(–) [22 ]. The percentage of cells with high mitochondrial potential, denoted as JC-1(+), was reported.
8
Molecular Cloning and Cell Culture Protocols
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Primers for cloning were obtained from the International DNA Technology (Coralville, IA). All chemicals for in vitro biophysical studies were purchased from Sigma Chemical Co. (St. Louis, USA). Reagents needed for cell culture were purchased from Life Technologies (Grand Island, NY), Expression vectors were obtained from Clontech (Mountain View, CA) and shRNA plasmids were from OriGene Technologies (Rockville, MD, USA).
Sodium oleate was purchased from Sigma Chemical Co. All primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Alexa Fluor-tagged secondary antibodies, BODIPY FL C16 and microscopy reagents were purchased from Molecular Probes, Inc. (Eugene, OR). Alkaline phosphatase conjugated secondary antibodies were procured from Santa Cruz and BCIP-NBT were from Merck (Darmstadt, Germany). The JC-1 kit was purchased from Cayman Chemicals (Ann Arbor, MI). DAPI, and CellLight Mitochondria-RFP were obtained from Life Technologies (Grand Island, NY).
Sodium oleate was purchased from Sigma Chemical Co. All primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Alexa Fluor-tagged secondary antibodies, BODIPY FL C16 and microscopy reagents were purchased from Molecular Probes, Inc. (Eugene, OR). Alkaline phosphatase conjugated secondary antibodies were procured from Santa Cruz and BCIP-NBT were from Merck (Darmstadt, Germany). The JC-1 kit was purchased from Cayman Chemicals (Ann Arbor, MI). DAPI, and CellLight Mitochondria-RFP were obtained from Life Technologies (Grand Island, NY).
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