Cells at a density of 1×106 were collected, resuspended, and added with 0.5 mL of JC-1 staining buffer (C2006, Beyotime, China), then incubated for 20 min in the incubator. By centrifuging at 4 °C (3 min, 400 g), the precipitate was collected and resuspended with JC-1 staining buffer. The Fluorescence was analyzed by NovoCyte software (ACEA Biosciences Inc., USA).
Jc 1 staining buffer
Manufactured by Beyotime
Sourced in China
JC-1 staining buffer is a laboratory reagent used in cell biology and biochemistry research. The buffer is designed to facilitate the staining of mitochondria with the fluorescent dye JC-1, which is commonly used to assess mitochondrial membrane potential.
Automatically generated - may contain errors
Lab products found in correlation
Novocyte software, by Agilent Technologies (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Jc 1 fluorescent probe kit, by Beyotime (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Flow cytometry, by Beyotime (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Jc 1 dyeing working solution, by Beyotime (1 mentions)
Jc 1 fluorescent probe, by Beyotime (1 mentions)
Flow cytometry, by BD (1 mentions)
Jc 1 dye, by Beyotime (1 mentions)
Fv1000, by Olympus (1 mentions)
10 protocols using jc 1 staining buffer
1
JC-1 Mitochondrial Membrane Potential Assay
2
Mitochondrial Membrane Potential Analysis
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The mitochondrial transmembrane potential was detected by adopting 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) staining. Briefly, the cells treated as indicated were incubated with a staining solution (Beyotime, Shanghai, China) containing JC-1 probe at 37 °C for 20 min in the dark. The mixture was then rinsed twice with JC-1 staining buffer (Beyotime, Shanghai, China). Images were observed under the fluorescence microscope (Olympus Optical Co., Ltd., Tokyo, Japan).
3
Mitochondrial Membrane Potential Analysis
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Mitochondrial membrane potential (MMP) was analyzed using a JC-1 fluorescent probe kit (Beyotime Institute of Biotechnology, Haimen, China). The lipophilic and cationic fluorescent dye, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazole carbocyanine iodide), is capable of selectively entering mitochondria, forming aggregates in mitochondria and emitting red fluorescence at high MMP. However, in low MMP, JC-1 cannot enter mitochondria and forms monomers that emit green fluorescence. The ratio of red to green fluorescence provides an estimate of change in MMP. The aggregates/monomer ratio was quantified at an excitation wavelength of 524 nm, emission wavelength of 590 nm (monomer, excitation at 490 nm and emission at 530 nm). HepG2 cells or L02 cells were co-cultured with SWNHs for 48 h in a 6-well plate and then treated with JC-1 (5 µg/ml) at 37°C for 30 min. Then, the cells were washed with JC-1 Staining Buffer (Beyotime Institute of Biotechnology) for three times. The fluorescence intensity was immediately measured using a fluorescence microscope at a magnification of ×200 (IX71; Olympus Corporation, Tokyo, Japan). The intensity of green and red fluorescence was measured using Image-Proplus 6.0 software (Media Cybernetics, Rockville, MD, USA).
4
Mitochondrial Membrane Potential Assay
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The positive control was treated with CCCP (10 mM) at 1:1,000 to medium at 37 °C for 20 min. The medium was aspirated, and 1 ml of medium and 1 ml of JC-1 working reagent (Beyotime, China) were added, followed by incubation at 37 °C for 20 min. Subsequently, the cells were washed with JC-1 staining buffer (Beyotime, China). An aliquot of 1 ml of medium was added to each well of the plate, and the absorbance was measured at 490 and 525 nm using a multifunctional microplate reader (Infinite200 PRO, TECAN, Switzerland).
5
Evaluating Cellular Stress Response Markers
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To evaluate ROS levels, kidney tissues were rinsed with PBS, resuspended in erythrocyte lysate, neutralized with an equal volume of PBS, centrifuged, and resuspended in PBS. ROS levels were detected using flow cytometry according to the manufacturer’s instructions (Beyotime, Shanghai, China).
To evaluate apoptosis, 1 × 106 cells were centrifuged and resuspended in 200 μL of PBS. 10 μL Annexin V-FITC and 10 μL PI (BD, New Jersey, USA) were gently mixed and incubated at 4 °C for 30 min in the dark. After the addition of 300 μL of PBS, the cells were subjected to flow cytometry.
To evaluate the mitochondrial membrane potential, 1 × 106 cells were resuspended in cell culture medium (0.5 mL) and incubated with JC-1 staining solution (0.5 mL) for 20 min. The cells were then centrifuged and resuspended in JC-1 staining buffer (Beyotime, Shanghai, China). Mitochondrial membrane potential was detected by flow cytometry.
To evaluate apoptosis, 1 × 106 cells were centrifuged and resuspended in 200 μL of PBS. 10 μL Annexin V-FITC and 10 μL PI (BD, New Jersey, USA) were gently mixed and incubated at 4 °C for 30 min in the dark. After the addition of 300 μL of PBS, the cells were subjected to flow cytometry.
To evaluate the mitochondrial membrane potential, 1 × 106 cells were resuspended in cell culture medium (0.5 mL) and incubated with JC-1 staining solution (0.5 mL) for 20 min. The cells were then centrifuged and resuspended in JC-1 staining buffer (Beyotime, Shanghai, China). Mitochondrial membrane potential was detected by flow cytometry.
6
Mitochondrial Membrane Potential Assay
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After the drug intervention, the cells were washed once with cold PBS, 1 mL cell culture medium, and 1 mL JC-1 staining buffer (Beyotime, Shanghai, China, C2003S). The cells were then added according to the kit instructions, mixed, and incubated for 20 min at 37°C in an incubator. The supernatant was discarded, and the JC-1 staining buffer was washed twice. A 2 mL cell culture medium was added, and the fluorescence signal was observed under a fluorescence microscope.
7
Mitochondrial Membrane Potential Assay
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MSCs were washed with PBS, and JC-1 dyeing working solution (Beyotime) mixed with fresh medium was added. The cells were incubated at 37°C for 20 min in a cell incubator. After incubation at 37°C, the supernatant was aspirated and the cells were washed twice with JC-1 staining buffer (Beyotime). Afterward, 2 ml of cell culture medium was added, and the results were observed under a fluorescence microscope. Relative degrees of mitochondrial polarization were quantified by measuring the red-shifted JC-1 aggregates and the green-shifted monomers.
8
Mitochondrial Membrane Potential Assay
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The ΔΨm was determined using an enhanced mitochondrial membrane potential detection kit (JC-1) (Beyotime, Shanghai, China). In short, 5 × 105 cells were resuspended in 0.5 mL cell culture medium; then, 0.5 mL JC-1 staining medium with JC-1 fluorescent probe (Beyotime, Shanghai, China) was added and mixed. After incubation at 37 °C for 20 min, the cells were harvested by centrifugation at 600× g for 3 min at 4 °C and then washed twice with JC-1 staining buffer (Beyotime, Shanghai, China). The cell pellets were resuspended in 0.5 mL JC-1 staining buffer. The fluorescence was read at the ex/em wavelength of 485/590 nm.
9
Evaluating Mitochondrial Depolarization in U251 Cells
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U251 (4 × 105 cells/well) cells were seeded in 6-well plates and then treated with 2 μM sorafenib and 100 μM TMZ at the indicated times. The control was cultured with medium containing an appropriate amount of DMSO. The cells were collected and gently resuspended in cell culture medium. Then, the corresponding volume of JC-1 dyeing solution and mix were added and the cells were incubated at 37°C for 20 min. Subsequently, the cells were centrifuged at 600 g at 4°C for 3 min. Then, the supernatant was discarded, 1 × JC-1 staining buffer (Beyotime Biotech, Nanjing, China) was added to resuspend the cells, and the cells were centrifuged at 600 g at 4°C for 3 min. The supernatant was discarded, and then 1 × JC-1 staining buffer was added to resuspend the cells, which were analyzed by flow cytometry (Becton-Dickinson, Franklin Lakes, NJ, United States). Mitochondrial depolarization was evaluated by detecting the excitation wavelengths of JC-1 (488 nm), and J-aggregate forms were assessed at 529 and 590 nm.
10
Assessing Mitochondrial Membrane Potential in Osteoblasts
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The collapsed MMP in osteoblasts was detected by flow cytometry following JC-1 dye staining (Beyotime-biotechnology, China). Briefly, after treatment as described above, the harvested MC3T3-E1 cells were resuspended in a staining solution, which were prepared by admixing serum-free a-MEM (500 µL) and JC-1 staining fluid (500 µL). Subsequently, osteoblasts were incubated with the staining solution for 20 min at 37 °C in the dark. After incubation, osteoblasts were washed thrice with the JC-1 staining buffer (Beyotime Institute of Biotechnology, Jiangsu, China), and resuspended in 500 µL of cell culture medium prior to analysis by flow cytometry. Finally, the ratio of red fluorescence intensity to green fluorescence intensity was calculated, and the results were used to evaluate the change in MMP for each sample (Ding et al., 2012 (link)).
In order to evaluate the change in MMP in situ, osteoblasts were loaded with JC-1 dye after treatment with FAC, as described above (Ding et al., 2012 (link)). Aggregated JC-1 (red fluorescence) and monomeric JC-1 (green fluorescence) levels were observed using laser scanning confocal microscopy (Olympus FV1000; Olympus, Tokyo, Japan).
In order to evaluate the change in MMP in situ, osteoblasts were loaded with JC-1 dye after treatment with FAC, as described above (Ding et al., 2012 (link)). Aggregated JC-1 (red fluorescence) and monomeric JC-1 (green fluorescence) levels were observed using laser scanning confocal microscopy (Olympus FV1000; Olympus, Tokyo, Japan).
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