MMP (ΔΨm) was monitored using Rhodamine 123 (Ex/Em = 485/535 nm; Sigma-Aldrich Co., St. Louis, MO, USA), a fluorescent, cell-permeable, cationic dye that preferentially enters into mitochondria, which typically have highly negative MMP (∆Ψm). Depolarization of MMP (∆Ψm) results in the loss of Rhodamine 123 from the mitochondria and reduces the intracellular fluorescence intensity of this dye [34 (link),35 (link)]. In brief, 1 × 106 cells in 60 mm culture dishes (BD Falcon) were pretreated with each caspase inhibitor (15 μM) for 1 h and then treated with 800 μM PG 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 FACStar flow cytometer (BD Sciences). Rhodamine 123-negative cells indicated the loss of MMP (∆Ψm) in lung cancer cells. MMP (ΔΨm) levels in cells, except for Rhodamine 123-negative cells, were expressed as percentages compared with control cells.
Rhodamine 123
Manufactured by BD
Sourced in United States
Rhodamine 123 is a fluorescent dye commonly used in laboratory applications. It is a cationic dye that exhibits green fluorescence when excited by light. Rhodamine 123 is often used as a fluorescent label or indicator in various cell biology and biochemical assays.
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Lab products found in correlation
Rhodamine 123, by Merck Group (11 mentions)
60 mm culture dishes, by BD (5 mentions)
Facstar flow cytometer, by BD (2 mentions)
Accuri c6 flow cytometry, by BD (2 mentions)
12 well culture plate, by BD (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Floid cell imaging station, by Thermo Fisher Scientific (2 mentions)
Rhodamine 123, by Thermo Fisher Scientific (2 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
Plga 50 50, by Merck Group (1 mentions)
β actin clone ac 74, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Bax antibody, by Santa Cruz Biotechnology (1 mentions)
Bcl 2 antibody, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Polyvinyl alcohol (pva), by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Epigallocatechin gallate (egcg), by Merck Group (1 mentions)
Cytochrome c, by Cell Signaling Technology (1 mentions)
16 protocols using rhodamine 123
1
Mitochondrial Membrane Potential Analysis
2
Measuring Mitochondrial Membrane Potential
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MMP (∆Ψm) levels were measured using JC-1 (Enzo Life Sciences; Ex/Em = 515 nm/529 nm) and rhodamine123 (Sigma-Aldrich Co.; Ex/Em = 485 nm/535 nm) dyes. In brief, 5 × 104 cells in a 12-well culture plate (BD Falcon) were incubated with the indicated concentrations of ATO and VPA alone and in combination for 72 h. Cells were washed twice with PBS and incubated with 10 μg/mL JC-1 or 0.1 μg/mL rhodamine 123 at 37 °C for 30 min. To stain nuclei, cells were incubated with 500 nM 4′, 6′-diamidino-2-phenylindole (DAPI, Life Technologies, Ex/Em = 358 nm/461 nm) at 37 °C for 30 min. After incubation with JC-1 and DAPI, cells were washed twice with PBS, and images were captured by fluorescence microscopy (FLoid® Cell Imaging Station, Life Technologies) at 400× magnification. Green fluorescence indicates lower MMP (∆Ψm), and red fluorescence indicates higher MMP (∆Ψm). The intensity of rhodamine 123 staining was determined by Accuri C6 flow cytometry (BD Sciences). Rhodamine 123 negative cells indicate the loss of MMP (ΔΨm).
3
Evaluating Mitochondrial Membrane Potential
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MMP (ΔΨm) was evaluated using the fluorescent dye rhodamine 123 (Ex/Em = 485/535 nm; Sigma-Aldrich), which is a cell-permeable cationic dye that preferentially enters into mitochondria because of their typical highly negative MMP (∆Ψm). Depolarization of MMP (∆Ψm) leads to the loss of rhodamine 123 from mitochondria and reduces the dye’s intracellular fluorescence intensity. Briefly, 1 × 106 cells in 60 mm culture dishes (BD Falcon) were incubated with the designated doses of auranofin 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 FACStar flow cytometer (BD Sciences). The absence of rhodamine 123 from cells indicates a loss of MMP (∆Ψm).
4
PLGA-based Nanoformulations for Cancer Therapy
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PLGA 50:50 (molecular weight 40–75 kDa), polyvinyl alcohol (molecular weight 30 kDa), 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT), TF, EGCG, CDDP, propidium iodide, and β-actin (clone AC-74) were purchased from Sigma-Aldrich (St Louis, MO, USA). Caspase-3, caspase-9, cytochrome C, p-NF-κB, p-IκBα, p53, and Bcl-2 antibodies were sourced from Cell Signaling Technology (Beverly, MA, USA) while Bax antibody was obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). The rabbit anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies were obtained from Bangalore Genei (Bangalore, India). The polyvinylidene fluoride membrane was obtained from Millipore (Bedford, MA, USA). Caspase-3 inhibitor z-DEVD-fmk was purchased from Calbiochem (Boston, MA, USA). Fetal bovine serum, Dulbecco’s Modified Eagle’s Medium, and Roswell Park Memorial Institute medium were supplied by Invitrogen (Invitrogen, Carlsbad, CA, USA) and the antibiotics by Gibco (Lifetech, Karlsruche, Germany). 2′,7′-dichlorofluorescein diacetate (DCF-DA) and rhodamine 123 from BD Pharmingen (San Diego, CA, USA) were used. Other chemicals used were of analytical grade and sourced locally.
5
Visualizing Bile Acid and MDR1 Transport
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The bile acid conjugate CLF has been used for visualisation of bile acid transportation across liver tissue3 (link),5 (link),6 ,25 (link), in particular as regulated by MRP2 whereas the activities of MDR1 was assessed by rhodamine 123 efflux8 (link),9 (link),19 (link). Independently, cysts were washed and incubated separately with 5 μM CLF (BD, Japan) and 100 μM rhodamine 123 (BD) in the free serum-culture medium, at 37 °C within 30 min. Subsequently, cysts were rinsed twice with fresh culture medium. The fluorescence dye accumulation was then recorded using a confocal microscope (Olympus, Japan) employing FITC (495–519 nm) for CLF and rhodamine green (502–527 nm) emission spectra.
6
Detecting MOMP via Rho-123 Assay
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For the detection of MOMP, the rhodamine 123 (Rho-123) assay was performed. For this, 1.5 × 105 cells/well were seeded in 6-well plates and grown overnight in 5% CO2 at 37 °C. Then cells were treated with the immunotoxin hD7-1(VL-VH)-PE40 or ABT-737 for 4, 24, or 48 h. 4 mg/mL cycloheximide was used as positive control. After that cells were trypsinized and centrifuged (430 g, 3 min). The pellet was resuspended in 10 µL PBS + 3% FCS + 0.1% sodium azide and incubated with 25 µL rhodamine 123 (10 µg/mL, Merck). After incubation for 30 min at 37 °C, cells were washed three times with PBS and resuspended in 200 µl PBS + 1 µg/mL propidium jodide (Merck). Decreasing fluorescence intensity of rhodamine 123 as a sign for MOMP was detected using a FACSCalibur flow cytometer and the software CellQuest Pro (BD Biosciences).
7
Measuring Mitochondrial Membrane Potential
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MMP (ΔΨm) levels were measured using rhodamine 123 (Sigma-Aldrich Co.; Ex/Em = 485 nm/535 nm) and JC-1 dyes (Enzo Life Sciences ; Ex/Em = 515 nm/529 nm). Briefly, 5 × 104 cells in 12 well culture plate (BD Falcon) were incubated with the indicated concentration of SAHA for 24 hours. Cells were washed twice with PBS and incubated with 0.1 μg/ml rhodamine 123 or 10 μg/ml JC-1 at 37°C for 30 min. For staining nucleus, cells were incubated with 500 nM 4′, 6′-diamidino-2-phenylindole (DAPI, Life Technologies, Ex/Em = 358 nm/461 nm) at 37°C for 30 min. The intensity of rhodamine 123 staining was determined by Accuri C6 flow cytometry (BD Sciences). The negative staining of rhodamine 123 from cells indicated the loss of MMP (ΔΨm) in cells. After incubation with JC-1 and DAPI, cells were washed three times with PBS and images were collected by using a fluorescence microscope (FLoid® Cell Imaging Station, Life Technologies) in × 400 magnification. Green fluorescence indicates a monomer at low ΔΨm and red fluorescence presents high ΔΨm.
8
Mitochondrial Membrane Potential Assay
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Rhodamine 123 was used to monitor the mitochondrial membrane potential. Cells were grown on 0.5% glucose and harvested in the log-phase and incubated with 50 nM Rhodamine123 (Invitrogen) for 30 min at 37°C. The fluorescence intensity of the cells stained with Rhodamine123 was analysed using a flow cytometer (BD Accuri™ C6 Plus) as described previously (Drakulic et al., 2005 (link)).
9
Measuring P-Glycoprotein Function Using Rhodamine-123 Efflux
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rhodamine-123 efflux was used to detect the P-gp function of cells. Cells were cultured in 6-well plates; at 70-80% confluence, rhodamine-123 (Sigma) was added to the cells at a final concentration of 0.25 µg/ml and incubated at 37°C for 1 h. Cells were washed 3 times with PBS at 4°C and re-suspended at 5-10 × 10 5 cells/ml in PBS.
rhodamine-123 fluorescence was analyzed using a FACStar flow cytometer (BD Biosciences, CA, USA) equipped with an argon laser. The blast population was gated by forward and side scatter characteristics. rhodamine-123 fluorescence of 10,000 cells was measured logarithmically through a 530-nm bandpass filter at an excitation wavelength of 488 nm. Cells that were not incubated with rhodamine-123 served as a D r a f t negative control. rhodamine-123 efflux was measured by counting cells in the M1 region of the plot and was expressed as a percentage. The higher the percentage of cells in the M1 region, the greater the cellular rhodamine-123 efflux and P-gp function.
rhodamine-123 fluorescence was analyzed using a FACStar flow cytometer (BD Biosciences, CA, USA) equipped with an argon laser. The blast population was gated by forward and side scatter characteristics. rhodamine-123 fluorescence of 10,000 cells was measured logarithmically through a 530-nm bandpass filter at an excitation wavelength of 488 nm. Cells that were not incubated with rhodamine-123 served as a D r a f t negative control. rhodamine-123 efflux was measured by counting cells in the M1 region of the plot and was expressed as a percentage. The higher the percentage of cells in the M1 region, the greater the cellular rhodamine-123 efflux and P-gp function.
10
Mitochondrial Membrane Potential Measurement
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The MMP (ΔΨm) was monitored using rhodamine 123 (Sigma-Aldrich Co.; Ex/Em = 485/535 nm), which is a fluorescent, cell-permeable, and cationic dye that favorably enters the mitochondria with high negative MMP (∆Ψm). Depolarization of MMP (∆Ψm) results in the loss of rhodamine 123 from the mitochondria and reduction of the intracellular fluorescence intensity of this dye, as previously described38 (link). Briefly, 1 × 106 cells in 60-mm culture dishes (BD Falcon) were pretreated with 2 mM NAC or 10 μM BSO for 1 h and then treated with PG 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. The intensity of rhodamine 123 staining was determined using a FAC Star flow cytometer. rhodamine 123-negative (−) cells represent HPF cells with MMP (∆Ψm) loss. MMP (ΔΨm) levels in cells without MMP (ΔΨm) loss were expressed as a percentage of levels in control cells.
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