Mitochondrial membrane potential (Δψm) was measured by flow cytometry using a fluorescent cationic dye, DePsipher, according to the vendor’s protocol (DePsipher, R&D System Inc.), as described previously [19 (link),20 (link),21 (link)]. The dye readily enters healthy mitochondria and fluoresces red in its multimeric form. However, in apoptotic cells, the mitochondrial membrane potential collapses, and the dye cannot enter the mitochondria and remains in the cytoplasm in its green fluorescent monomeric form.
Depsipher
Manufactured by R&D Systems
Sourced in United States
DePsipher is a versatile laboratory instrument designed for the analysis and detection of various biomolecules. It utilizes advanced spectroscopic techniques to provide accurate and reliable data. The core function of DePsipher is to enable the identification and quantification of target analytes in complex samples.
Automatically generated - may contain errors
7 protocols using depsipher
1
Measuring Mitochondrial Membrane Potential
2
Mitochondrial Membrane Potential in Rin-5F Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial membrane potential was measured using a fluorescent cationic dye, DePsipher ™ (R & D Systems, MN, United States)in control and LPS and/or AZD-treated Rin-5F cells, and quantitated by flow cytometry as described before (Alnahdi et al., 2019a (link)). The dye which could not access the transmembrane space due to low membrane potential remained in its green fluorescent form and this was measured using a flow cytometer (Becton Dickinson FACSCanto II).
3
Apoptosis Detection in Rat Cardiomyocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ventricular myocytes from 1 to 3 day-old Sprague-Dawley rats were enzymatically dissociated, purified by Percoll gradient centrifugation and pre-plating, and cultured as described (Oh et al., 2003 (link)). To detect hypodiploid DNA, flow cytometry was performed (XL or LSRII, BD Biosciences), using propidium iodide to measure DNA content and sampling > 5000 cells for each histogram. Myocyte identity was confirmed using FITC-conjugated MF-20 antibody to sarcomeric MyHC (R&D Systems). To detect dissipation of mitochondrial membrane potential (ΔΨm), cells were incubated for 60 min in 5 μg ml-1 5, 5′, 6, 6’-tetrachloro-1, 1’, 3, 3′-tetraethylbenzimidazolyl carbocyanine iodide (DePsipher; R&D Systems). When ΔΨm is intact, mitochondrial uptake and aggregation of the dye result in red fluorescence; when ΔΨm is disturbed, the dye diffuses to the cytoplasm and reverts to its monomeric green form. Myocyte identity was confirmed using mouse antibody to sarcomeric tropomyosin (Sigma-Aldrich) conjugated with Texas Red-X (Molecular Probes).
4
Mitochondrial Membrane Potential Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mitochondrial membrane potential was measured according to the manufacturer’s instructions (DePsipher; R&D Systems, Inc., Minneapolis, Minnesota) by fluorescent microscopy using a fluorescent cationic dye, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). 5 ×104 cells/well were cultured in 12-well plates for 48 hours in the presence of increasing concentrations of TXA with medium (0 mg/ml, 1 mg/ml, 50 mg/ml, or 100 mg/ml). The cells were then washed with PBS, and reaction buffer was added with DePsipher (5 µl per sample) and stabilizer solution. The cells were incubated at room temperature, in the dark, for a minimum of 15 minutes. The cells were analyzed within one hour by fluorescent microscopy using a fluorescein long-pass filter (EVOS FL Auto 2 Imaging System; Thermo Fisher Scientific, Waltham, Massachusetts). Using this system, cells undergoing apoptosis should appear green (585/590 nm), while healthy cells appear red (510/527 nm). Four corner random field images were saved and the number of apoptotic (green) and live (red) cells counted per image. The percentage of apoptotic cells as a proportion of total cells was calculated for each image and the mean percentage calculated for each sample.
5
Mitochondrial Membrane Potential Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Macrophage cells (2–6 ×106 cells/well) were treated with LPS/ASA with or without NAC as described above. The mitochondrial membrane potential was measured according to the vendor's protocol (DePsipher, R &D Systems Inc.) by flow cytometry using a fluorescent cationic dye as described before [13] (link). DePsipher has the property of aggregating upon membrane polarization forming an orange-red fluorescent (absorption/emission 585/590 nm) compound. If the membrane potential is reduced, the dye cannot access the transmembrane space and remains in its green fluorescent (510/527 nm) monomeric form.
6
Mitochondrial Membrane Potential Measurement
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial membrane potential was measured in cells after treatment with varying concentrations of glucose/palmitic acid for 24 h using a fluorescent cationic dye, DePsipher TM (R&D System Inc.), as described previously [25 (link),26 (link),27 (link)]. If the membrane potential is reduced, the dye cannot access the transmembranee space and remains in its green fluorescent form.
7
Mitochondrial Function Assay in Cytarabine-Treated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial membrane potential was assessed using DePsipher (R&D Systems), a lipophilic cation that has the property of aggregating upon membrane polarization, forming an orange-red fluorescent compound. If the potential is disturbed, the dye cannot access the transmembrane space and remains or reverts to its green monomeric form. The cells were stained with DePsipher as described by the manufacturer, and the green monomer and red aggregates were detected using a FACSCalibur flow cytometer and Cell Quest Pro software. The results are presented as a green/red fluorescence ratio (FL1/FL2, arbitrarily set to 1 in control samples), the increase of which reflects mitochondrial depolarization. The production of superoxide radical was measured using a superoxide-selective fluorochrome dihydroethidium (DHE) (Life Technologies, Carlsbad, CA). DHE (20 μM) was incubated with the cells for the last 30 min of the treatment and the mean intensity of red fluorescence (FL2), corresponding to superoxide levels, was determined using a FACSCalibur flow cytometer and Cell Quest Pro software. Based on preliminary time-dependence experiments, both mitochondrial membrane potential and superoxide production were analyzed after 24 h of treatment with cytarabine.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!