Mitochondrial membrane potential was tested using the JC-1 mitochondrial membrane potential detection kit (KeyGEN, KGA602, China). Aggregated JC-1 emits red fluorescence, whereas the monomorphic form emits green fluorescence. In impaired mitochondria, with a depolarized mitochondria membrane potential, most JC-1 is in monomeric form, giving a low red-to-green fluorescence ratio [24 (link)]. Pretreated PC12 cells were washed and incubated with JC-1 (2 μg/ml) for 15 min at 37°C following the manufacturer’s instructions. Immediately after washing cells twice with incubation buffer, cell fluorescence emission was measured using a flow cytometer (Merck, Amnis Flowsight, USA).
Jc 1 mitochondrial membrane potential detection kit
Manufactured by Keygen Biotech
Sourced in China
The JC-1 mitochondrial membrane potential detection kit is a laboratory instrument used to measure the mitochondrial membrane potential in cells. It works by utilizing the fluorescent dye JC-1, which exhibits potential-dependent accumulation in mitochondria. The kit provides the necessary reagents and protocols to perform this analysis.
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Lab products found in correlation
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi, by Merck Group (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Annexin 5 conjugated fitc apoptosis detection kit, by Keygen Biotech (2 mentions)
Macs flow cytometer, by Miltenyi Biotec (1 mentions)
Trypsin, by Merck Group (1 mentions)
Macsquantify software, by Miltenyi Biotec (1 mentions)
Enhanced chemiluminescence ecl kit, by Cytiva (1 mentions)
β actin, by Proteintech (1 mentions)
Secondary antibodies conjugated to horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Caspase 3 activity detection kit, by Keygen Biotech (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Cyclin b1, by Cell Signaling Technology (1 mentions)
3 4 5 dimethylthiazole 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Antibodies against caspase 3, by Cell Signaling Technology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
6 protocols using jc 1 mitochondrial membrane potential detection kit
1
Assessing Mitochondrial Membrane Potential
2
JC-1 Mitochondrial Membrane Potential Assay
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According to the instructions of the JC-1 mitochondrial membrane potential detection kit (KeyGen Biotech, China), the treated cells were incubated with the dye mixture at 37 °C for 30 min and then washed with PBS three times. The cells were observed under a laser confocal microscopy and recorded by a random typical visual field. The red:green fluorescence ratio was calculated by ImageJ software (version 1.4.3.67).
3
Mitochondrial Membrane Potential Analysis
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Quantitative changes in ΔΨm at the early stage of cell apoptosis were measured using a J aggregate-forming lipophilic cation-1 (JC-1) Mitochondrial Membrane Potential Detection kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions. Cells were treated with 4, 8, or 12 µM LFG-500 for 24 h, then digested with trypsin (Sigma-Aldrich; Merck Millipore) and centrifuged at 367 × g for 5 min at 4°C. The harvested cells were washed once with PBS and incubated with 10 µM JC-1 dye in incubation buffer at 37°C for 20 min in the dark. Subsequently, the cells were washed twice with pre-chilled buffer solution. Relative fluorescence intensities of the cells were monitored using a MACS Flow Cytometer (Miltenyi Biotec GmbH) with MACSQuantify Software (Miltenyi Biotec GmbH; software version 2.4).
4
Proanthocyanidins Modulate Apoptosis Signaling
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Antibodies against caspase-3, caspase-9, Bcl-2 were purchased from Cell Signaling Technology (Denver, MA). Antibodies against MMP-9, NFκB, IκBα and β-actin were obtained from Protein Tech Group, Inc. (Chicago, USA). Antibodies against phosphorylated IκBα, ERK1/2, p38 and JNK were obtained from Sangon Biotech, China. The enhanced chemiluminescence (ECL) kit was purchased from Amersham Life Science, Inc. (United States). The Annexin V-conjugated FITC apoptosis detection kit and JC-1 mitochondrial membrane potential detection kit were purchased from NanJing KeyGen Biotech Co., Ltd (Nanjing, China). Transwells were purchased from BD Biosciences (San Jose, USA). MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide) and DAPI (2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride) were obtained from Sigma Chemical Co. (St. Louis, MO). PMBPs powder was purchased from Shaanxi Sciphar Hi-tech Industry Co., Ltd (Shanxi, China). It contained approximately 95% proanthocyanidins, and stable for at least two years at 4°C.
5
Investigating Apoptotic Pathways in Cells
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Antibodies specific for Bcl-2, Bak-1 and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Secondary antibodies conjugated to horseradish peroxidase were purchased from Thermo Fisher Scientific, Inc. (New York, NY). The Annexin V-conjugated FITC apoptosis detection kit was obtained from BD Pharmaceuticals (Franklin Lakes, NJ). The JC-1 mitochondrial membrane potential detection kit and Caspase-3 activity detection kit were purchased from NanJing KeyGen Biotech Co., Ltd. (Nanjing, China), and the in situ cell death detection kit was purchased from Roche Diagnostic Corporation (Indianapolis, IN). MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide), PI (Propidium iodide) and DAPI (2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride) were obtained from Sigma Chemical Co. (St. Louis, MO). The GSPs were purchased from Jianfeng Natural Product R&D Co., Ltd. (Tianjin, China).
6
Scutellarin-Induced Apoptosis in Cells
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Antibodies against caspase-3, caspase-9, Bcl-2, Bax, Cdc2, cyclin B1, β-actin and γH2AX were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). The enhanced chemiluminescence (ECL) kit was purchased from Amersham Life Science, Inc. (Arlington Heights, IL, USA). The Annexin V-conjugated FITC apoptosis detection kit and JC-1 mitochondrial membrane potential detection kit were purchased from NanJing KeyGen Biotech Co., Ltd. (Nanjing, China). The Comet Assay kit was from NanJing KeyGen Biotech Co., Ltd. (Nanjing, China). 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 46-diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Scutellarin (>98%) powder was purchased from Sichuan Best-Reagent Industry Co., Ltd. (Sichuan, China, lot no. B01146801) and dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was 0.1% in all groups and had no effect on cell viability. The chemical formula of Scutellarin is C21H18O12.
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