To measure the mitochondrial transmembrane potential, JC-1 dye (Sigma-Aldrich), a sensitive fluorescent probe, was used. Fluorescence microscopy with a 488-nm filter was used for the excitation of JC-1. Emission filters of 535 and 595 nm were used to quantify the population of mitochondria with green (JC-1 monomers) and red (JC-1 aggregates) fluorescence, respectively. Immunolabeling was examined by an Eclipse Ti-U microscope (Nikon) and BD FACS Verse flow cytometer.
Jc 1 dye
Manufactured by Merck Group
Sourced in United States
JC-1 dye is a fluorescent probe used in cell biology research. It is capable of selectively entering mitochondria and can be used to monitor changes in mitochondrial membrane potential.
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Lab products found in correlation
Puromycin, by Merck Group (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Polybrene, by Merck Group (3 mentions)
Eclipse ti u microscope, by Nikon (2 mentions)
Facsaria iiu, by BD (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
Dcfh da, by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Infinite m1000 pro, by Tecan (2 mentions)
Biocoat plates, by Corning (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Facsverse, by BD (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
37 protocols using jc 1 dye
1
Mitochondrial Membrane Potential Assessment
2
Evaluating Mitochondrial Membrane Potential
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The mitochondrial membrane potential of cells was evaluated using JC-1 dye (Sigma, St. Louis, MO) as previously reported [13 (link)]. Briefly, cells were treated with the IC50 and IC50/5 of the P. nigrum extract and the controls for 6 h and 12 h, as previously published [19 (link)] (Supplementary Materials and Methods). The cells were acquired on a FACSAria II-U (Becton Dickinson, BD, NJ, USA) and analyzed with FlowJo v10.8.1 software (BD Life Sciences) which calculated the red/green fluorescence ratios.
3
Cellular Stress Response Pathway Analysis
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DEX, polybrene, puromycin, JC-1 dye, MG-132, cycloheximide (CHX), and 3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) dye were obtained from Sigma-Aldrich Chemicals (St. Louis, MO). The following antibodies for p44/42 MAPK (Erk1/2, #9102), HO1 (#70081), NQO1 (#3187), Nrf2 (#12721), Keap1 (#8047), α-Tubulin (#2125), and Lamin B1 (#13435) as well as cleaved-poly (ADP-ribose) polymerase (PARP, #5625), cleaved-caspase-3 (#9664) were purchased from Cell Signaling Tech (Shanghai, China). The anti-GCLC antibody (ab55435) and the anti-adenine nucleotide translocase 1 (ANT-1) antibody (ab102032) were provided by Abcam (Shanghai, China). The following antibodies for PGK1 (sc-130335), cyclophilin-D (CyPD, sc-137136), and VDAC1 (sc-390996) were purchased from Santa Cruz Biotech Co. (Santa Cruz, CA). The reagents for cell culturing, including fetal bovine serum (FBS), DMEM, penicillin and streptomycin were obtained from Gibco-BRL Co. (Grand Island, NY). TRIzol and other RNA assay agents, Annexin V, propidium iodide (PI) and cell transfection reagents (Lipofectamine 2000 and others) were from Invitrogen Thermo-Fisher (Shanghai, China). mRNA primers were purchased from Genechem Co. (Shanghai, China). All viral constructs and sequences were also designed and provided by Genechem Co.
4
Mitochondrial Membrane Potential Assay
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Cells treated with poly (I:C) was collected and washed twice with cold PBS. The pipetted cell was re-suspended with PBS supplemented with 5 μM JC-1 dye (Sigma-Aldrich, USA). After incubation at 37 °C for 15 min, the cells were observed with a fluorescence microscope.
5
Measuring Mitochondrial Potential with JC-1
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To measure the mitochondrial transmembrane potential, JC-1 dye (Sigma-Aldrich), a sensitive fluorescent probe, was used. Fluorescence microscopy with a 488 nm filter was used for the excitation of JC-1. Emission filters of 535 and 595 nm were used to quantify the population of mitochondria with green (JC-1 monomers) and red (JC-1 aggregates) fluorescence, respectively. Immunolabeling was examined by an Eclipse Ti-U microscope (Nikon). A total of 10 fields-of-view were randomly selected for analysis.
6
Sperm Mitochondrial Activity Evaluation
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The sperm mitochondrial activity was investigated using, JC-1 dye (T4069, Sigma-Aldrich, USA). Briefly,
post-thawed semen specimens were centrifuged at 500 g
for 5 minutes. The supernatant was removed, and cell pellets were resuspended in phosphate-buffered saline (PBS)
at a final concentration of 1×106 cells/ml. Then, 1 µL of
JC-1 stock solution [200 μM dissolved in DMSO (D2650,
Sigma-Aldrich, USA)] was added into 1 ml of cell suspension, incubated at 37°C for 40 minutes in a dark place,
and cells were finally subjected to flow cytometry (11 (link)).
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The sperm mitochondrial activity was investigated using, JC-1 dye (T4069, Sigma-Aldrich, USA). Briefly,
post-thawed semen specimens were centrifuged at 500 g
for 5 minutes. The supernatant was removed, and cell pellets were resuspended in phosphate-buffered saline (PBS)
at a final concentration of 1×106 cells/ml. Then, 1 µL of
JC-1 stock solution [200 μM dissolved in DMSO (D2650,
Sigma-Aldrich, USA)] was added into 1 ml of cell suspension, incubated at 37°C for 40 minutes in a dark place,
and cells were finally subjected to flow cytometry (11 (link)).
7
Evaluating Mitochondrial Dysfunction in P. falciparum
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To determine the effect of ART, ARS, and CDRI-97/78 on the mitochondria of erythrocytic stages of P. falciparum, iRBCs were exposed with these drugs for 6 and 24 h and further mature stage enriched parasitized red blood cells were stained with 6 μM JC-1 dye (Sigma-Aldrich) for 20 min. in dark at 37°C. Cells were washed twice and resuspend in 0.5 ml assay buffer for acquiring the fluorescence using flow cytometer.
Acquisition was carried out using FACS-CELLQUEST software on FACS Calibur (BD Biosciences) and analysis of flow cytometeric data was performed using Flow Jo software (Tree star Inc., Ashland, OR). Dissipation in ΔΨm was measured by calculating the ratio between red and green fluorescence (i.e., 590/530 nm) (Gunjan et al., 2016 (link)).
Acquisition was carried out using FACS-CELLQUEST software on FACS Calibur (BD Biosciences) and analysis of flow cytometeric data was performed using Flow Jo software (Tree star Inc., Ashland, OR). Dissipation in ΔΨm was measured by calculating the ratio between red and green fluorescence (i.e., 590/530 nm) (Gunjan et al., 2016 (link)).
8
Evaluating Aflatoxin B1 and Saikosaponin T in HepG2 Cells
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Human hepatoma HepG2 cells lines were obtained from American Type Culture Collection (ATCC, Beijing, China). AFB1 (purity ≥ 98%), ST (purity ≥ 98%), DMSO, sulforhodamine B (SRB), TCA, H33258, DCFH-DA, DCF, rhodamine 123, JC-1 dye, and calf thymus DNA were purchased from Sigma–Aldrich (Shanghai, China). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin, HBSS, and phosphate-buffered saline (PBS) were purchased from Gibco Life Technologies (Shanghai, China). ATP assay kit, Annexin V-FITC cell apoptosis assay kit, and mitochondria membrane potential assay kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China). DAB was purchased from Genetech Inc. (Shanghai, China). All the antibodies for Caspase-3, p53, Bax and Bcl-2 were from Germany AbioB, LTM. (Shanghai, China).
9
Mitochondrial Membrane Potential Measurement
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CD4+ naïve T cells co-cultured with CD70-NC and CD70-KO C666 cells in lipid-depleted or normal culture medium after 3 days were stained with JC-1 dye (Sigma) for 20 mins in the dark at 37 °C, as per manufacturer’s instructions. T cells were washed twice with cold washing buffer and centrifuged at 800× g for 5 min. After the final wash, T cells were re-suspend in 0.5 ml cold washing buffer for acquiring and analyzing the PE (JC-1 aggregates) and FITC (JC-1 monomers) fluorescence by ACEA Novocyte Quanteon.
10
Sperm Viability and DNA Damage Assessment
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Glucose (G), sucrose (S), trehalose (T), l -ascorbic acid (L-As), dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), glycerol (GLY), methanol (MeOH), and JC-1 dye were purchased from Sigma-Aldrich (S. Louis, MO, United States). Phosphate buffered saline (PBS: Ca2+ and Mg free) was obtained from Life Technologies Ltd. (Paisley, UK). LysoTracker™ green DND-26, and the LIVE/DEAD® sperm viability kit were bought from Invitrogen Molecular Probes (Eugene, OR, United States). A comet assay® (single-cell gel electrophoresis) kit was purchased from Trevigen Inc. (Gaithersburg, MD, United States).
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