MMP (ΔΨm) was examined using a cell-permeable and cationic rhodamine 123 fluorescent dye (Sigma-Aldrich Co.; Ex/Em = 485/535 nm). Briefly, 1 × 106 cells in 60 mm culture dishes (BD Falcon) were incubated with Ebselen at the indicated concentrations (2–20 μM) for 24 h. Cells were washed twice with PBS and incubated with rhodamine 123 (0.1 mg/mL) at a concentration of 5 × 105 cells/mL at 37 °C for 30 min. Rhodamine 123 staining intensities were determined using a FAC Star flow cytometer (BD Sciences). Rhodamine 123-negative (−) cells designated MMP (ΔΨm) loss cells.
Rhodamine 123 fluorescent dye
Manufactured by Merck Group
Sourced in United States
Rhodamine 123 is a fluorescent dye used as a labeling agent in various biological and chemical applications. It exhibits green fluorescence when excited by a suitable light source. The dye is commonly used for staining mitochondria in living cells, as it selectively accumulates in these organelles. Rhodamine 123 can be used in flow cytometry, fluorescence microscopy, and other techniques that require a fluorescent marker.
Automatically generated - may contain errors
Lab products found in correlation
60 mm culture dishes, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Cinnamaldehyde, by Merck Group (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Flow cytometry, by BD (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
7 protocols using rhodamine 123 fluorescent dye
1
Mitochondrial Membrane Potential Assay
2
MMP Assessment Using Rho123 Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were plated at a density of 1×104 cells/well into 6-well plates, where they were cultured for 24 h. The cells were treated with various doses of PSPA (100, 300 and 500 µg/ml) for 48 h. A flow cytometry assay was employed to assess the MMP with Rhodamine123 fluorescent dye (Rho123; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The deteriorated Rho123 accumulation in the mitochondria, indicated the collapse of the MMP. The cells were incubated with a Rho123 working solution in the dark, for 30 min, at 37°C. The cells were then loaded and the fluorescent signals with an emission of 525 nm were applied on a FACSCalibur (Becton-Dickinson, Franklin Lakes, NJ, USA).
3
Evaluating Mitochondrial Membrane Potential
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial membrane potential (MMP) is closely linked to the intrinsic pathway of apoptosis and was evaluated by the method described previously [15 (link)]. Both cell lines were treated with SB as described previously and, immediately after detachment, were washed with PBS and used to measure mitochondrial membrane potential (∆ψm) using Rhodamine 123 fluorescent dye (Sigma-Aldrich, USA), which is taken by mitochondria of living cells. The cells were incubated with Rhodamine 123 (5 µM final) and prepared as a stock solution (1 mg/mL in ethanol) for 20 min at 37 °C. The supernatant was removed after pelleting the cells, and MMP was measured in 150 µL of PBS by flow cytometry with the excitation of Rhodamine 123 at 505 nm and emission at 535 nm. Data were obtained using a FACS Canto flow cytometer (Becton Dickinson Biosciences, USA), analyzed using FACS Diva software, and expressed as the mean fluorescence intensity of Rhodamine 123.
4
Mitochondrial Membrane Potential Assay
5
Rhodamine-123 Mitochondrial Membrane Potential
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rhodamine‐123 fluorescent dye (Sigma‐Aldrich, St. Louis, MO, USA) was employed to detect mitochondrial membrane potential. The culture medium containing different concentrations of LIG was added to treat for 2 hours, followed by SNP (0.75 mmol/L) co‐treatment for 24 hours. After each treatment, the cells were incubated with 2 mL of DMEM/F12 complete culture medium with 10 μmol/L Rhodamine‐123 for 30 minutes at 37°C. Then, the cells were rinsed twice with PBS, and fluorescence signals were imagined under an Olympus microscope. The mitochondrial membrane potential was measured using Image‐Pro Plus 6.0 software.
6
Cytotoxicity and Mitochondrial Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cinnamaldehyde, Triton® X-100, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), rhodamine 123 fluorescent dye, Bradford reagent, doxorubicin and dimethyl solphoxide (DMSO) were purchased from Sigma Aldrich (St Louis, MO, USA). Dulbecco’s modified eagle’s medium (DMEM), fetal bovin serum (FBS), and penicillin/streptomycin were supplied from Gibo (USA). Trypsin-EDTA was prepared from Bon Yakhteh (I.R. Iran).
7
Measuring Mitochondrial Membrane Potential
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mitochondrial membrane potential was monitored using Rhodamine-123 fluorescent dye (Sigma), a cell-permeable cationic dye, which preferentially enters into mitochondria due to the highly negative mitochondrial membrane potential (ΔΨm). Depolarization of ΔΨm results in the loss of Rhodamine-123 from the mitochondria and a decrease in intracellular fluorescence. After treatment, cells were harvested and washed with PBS. The cell pellets were then resuspended in 5 mL of fresh incubation medium containing 2.0 μM rhodamine 123 and incubated at 37°C for 20 min and then analyzed by flow cytometry (Becton Dickinson, San Jose, CA). Transfection MM cells were transiently transfected with control short interfering RNA (siRNA), ATF4 siRNA, myr-Akt construct using the Lipofectamine 2000 reagent (Life technologies) according to the manufacturer's protocol. Briefly, 4 μl of 20 μM siRNA or plasmid (10 ng) was mixed with 200 μl of Opti-MEM. Lipofectamine 2000 (4 μl) was diluted in 200 μl of Opti-MEM and incubated at room temperature for 5 min. After incubation, the diluted Lipofectamine 2000 was mixed with the diluted siRNA and further incubated for 20 min at room temperature. A total 400 μl of the siRNA/plasmid-Lipofectamine 2000 complex was applied to each well of the cultured cells at approximately 50%-70% confluence in 6-well microplates.
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!