HK-2 cells were seeded in 96 well plate (1 × 104 cells/well) overnight and treated with cisplatin, cisplatin + Nelumbonymphaea (NY), Paeonia suffruticosa (PS) for 30 min. ROS production assays was stained by DCFDA cellular ROS detection assay kit according to the manufacture instruction (Abcam, Cambridge, USA, ab113851). The mitochondrial transmembrane potential was determined with JC-1 dye staining according to the manufacture protocol (Abcam, Cambridge, USA, ab113850). The fluorescence of the JC-1 monomer (green) was read in fluorescence plate reader with excitation/emission setting at 485/535 (Spectramax i3, Molecular Devices. Sunnyvale, USA). Activity of caspases were determined using a colorimetric caspase-9, -3 assay kit (Abcam, Cambridge, USA, ab65608, ab39401, respectively). The assays were performed 60 pi-well plates (5 × 105 cells/well) by incubating cell lysis buffer. After lysis, we measured caspase assay kits according to the manufacture protocol. Apoptotic cell measures were conducted via Annexin V-FITC/PI staining. Adherent cells underwent the collection from centrifugation and then re-suspended in 500 μl 1 × binding buffer. The cells were stained with 5 μL Annexin V–FITC and 5 μl propidium iodide (PI) (50 μg/mL) and incubated at room temperature for 15 min in the dark. The cells were analyzed by a flow cytometry (Becton Dickinson FACS Vantage SE, Sanjose, USA).
Ab113850
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab113850 is a reagent used in research applications. It is an antibody that can be used to detect and quantify the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Nc1463153, by PerkinElmer (2 mentions)
Dm il led, by Leica (2 mentions)
Facsvantage se, by BD (1 mentions)
Dcfda cellular ros detection assay kit, by Abcam (1 mentions)
Spectramax i3, by Molecular Devices (1 mentions)
Total reactive oxygen species assay kit, by Thermo Fisher Scientific (1 mentions)
Axio imager a1 microscope, by Zeiss (1 mentions)
Fluorescence microscope, by Echo Medical Systems (1 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
880 airyscan microscope, by Zeiss (1 mentions)
Plan apochromat 63 1.4 oil dic m27 objective, by Zeiss (1 mentions)
I3x plate reader, by Molecular Devices (1 mentions)
Prism 9, by GraphPad (1 mentions)
Mitotracker red, by Olympus (1 mentions)
Facscanto plus software, by BD (1 mentions)
Mitotracker red, by Cell Signaling Technology (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
F 2500, by Hitachi (1 mentions)
Fluorescence plate reader, by BMG Labtech (1 mentions)
32 protocols using ab113850
1
Cisplatin-induced Apoptosis in HK-2 Cells
2
Mitochondrial Potential and ROS Assays
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The relative immunofluorescence of a JC-1 probes (#ab113850, Abcam) was applied to analyze the mitochondrial potential, based on a previous study 40 (link). Intracellular ROS levels were assessed with a Total Reactive Oxygen Species Assay Kit (#88-5930-74, ThermoFisher, Inc.) 41 (link).
3
Measuring Mitochondrial Membrane Potential
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Mitochondrial membrane potential was determined using a commercial JC-1 mitochondrial membrane potential assay kit (ab113850, Abcam, USA) following the manufacturer’s instructions. Briefly, cells were seeded at 1.5 × 104 cells per well in a dark 96-well plate and allowed to attach overnight. Cells were washed with 1x dilution buffer and incubated with 10 μg/mL JC-1 solution for 10 min at 37 °C in the dark. After washing the plate twice with 1x dilution buffer, the results were obtained under an Axio Imager A1 microscope (Carl Zeiss, Germany).
4
Measuring Mitochondrial Membrane Potential in Endometrial Cancer Cells
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MMP were measured using an MMP assay kit (ab113850; Abcam, Cambridge, MA, USA), according to the manufacturer’s instructions. For this, endometrial cancer cells were seeded onto 96-well black plates ((#NC1463153, Perkin Elmer, Waltham, MA, USA) at a density of 1.5 × 104 cells/well. After an overnight incubation, cells were treated with SHetA2 or vehicle for the next 24 h. Afterwards, cells were washed with 1× dilution buffer and incubated with 20 µM JC-1 fluorescent dye (5′,6′,-tetrachloro-1,1′,3,3′ tetraethyl-benzimidazolyl carbocyanine iodide) for 10 min at 37 °C followed by two times washing with 1× dilution buffer. The fluorescence intensities for J-aggregates and J-monomers was measured at excitation and emission wavelengths of 535 nm and 590 nm, and 475 nm and 530 nm, respectively. The ratios of J-aggregates and J-monomers were calculated.
5
Mitochondrial Membrane Potential Assay
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The MMP was analyzed using JC-1 fluorescent dye (5′,6′,-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolyl carbocyanine iodide) and an MMP assay kit (ab113850; Abcam, Cambridge, MA, USA), following the manufacturer’s protocol. Endometrial cancer cells (1.5 × 104 cells/well) were seeded onto a 96-well black plate ((#NC1463153, Perkin Elmer, Waltham, MA, USA) and allowed to adhere overnight followed by sulforaphane treatment as shown. Cells were then washed with 1X dilution buffer provided in the kit and probed with 20 uM JC-1 for 10 min at 37 °C. Subsequently, cells were washed twice with 1X dilution buffer and fluorescence intensity was determined for J-aggregates and J-monomers at excitation and emission wavelengths of 535 nm and 590 nm and 475 nm and 530 nm, respectively. The ratio of J-aggregates and J-monomers was calculated. A decrease in fluorescent intensity ratio is suggestive of Δψm. depolarization.
6
Mitochondrial Membrane Potential Assay
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The mitochondrial membrane potential (ΔΨm) was detected by using the mitochondrial membrane potential assay kit (ab113850; Abcam, Cambridge, MA). The 100 μM carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP, a potent mitochondrial membrane disruptor)‐treated group was experimentally designed as a positive control group. In brief, after drug treatment, cells were incubated with 100 μL/well fluorescent cationic dye 5,5,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimi‐dazoylcarbocyanine iodide (JC‐1) solution at 37°C for 10 min in the dark. Finally, the JC‐1 monomers and JC‐1 aggregates (green fluorescence for monomer, red fluorescence for aggregate) were detected by fluorescent microscopy with exciting light at 490 nm and 535 nm, respectively. Besides, we used a fluorescent microplate reader to record fluorescence at Ex/Em = 490/535 nm.
7
Measuring Mitochondrial Membrane Potential
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MMP was measured using JC-1 dye (Abcam, ab113850), according to manufacturer’s instructions. Briefly, cells were incubated with 5 μM JC-1 for 20 minutes, then cells were washed twice with the completed medium and fluorescence was detected by flow cytometry (BD Biosciences).
8
Mitochondrial Membrane Potential Assay
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The mitochondrial membrane potential (ΔΨm) was measured by JC-1 (ab113850, Abcam, UK). Differentiated 3T3-L1 cells were treated with 5-μm JC-1 for 20 min at 37°C. Then, the cells were washed with PBS. To label the mitochondria, 50 nM of MitoTracker (M-7510, Thermo Scientific) in media without FBS was added to cells, and they were incubated at 37°C for 1 h. The cells were then washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. They were observed with a fluorescence microscope (ECHO).
9
Mitochondrial Membrane Potential Imaging
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Mitochondrial membrane potential was conducted as per the manufacturer’s recommendations (Abcam, Cat #ab113850) and plotted as a ratio of aggregated JC-1 form/monomer JC-1 form, with higher ratios indicating more polarized membranes. JC-1 aggregates were imaged at a 618 nm emission and a 488 nm excitation, and JC-1 monomers at a 536 nm emission and 488 nm excitation in Ringer’s solution (in mM, 160 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 8 D‐Glucose) using a Plan‐Apochromat 63× 1.4 Oil DIC M27 objective and a Zeiss 880 Airyscan microscope at RT. Images were analyzed using ImageJ software.
10
Mitochondrial Membrane Potential Analysis
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Mitochondrial membrane potential was assessed by using the JC-1 probe assay (ab113850, Abcam). HepG2 cells were plated at 10,000 cells per well in black, clear-bottom 96-well plates. Cells were stimulated for 1, 6, 12, and 24 h before addition of 1 mM JC-1 solution in the corresponding stimulation media. Cells were incubated for 10 min in the dark at 37°C and washed twice with PBS. Stained cells were imaged on the i3x Plate reader (Molecular Devices) with 535 nm excitation and 590 nm emission for aggregates and 475 nm excitation and 530 nm emission for monomers. Statistics were carried out on Prism 9.1 (Graphpad).
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