For fluorescence analysis of mitochondrial morphology, a mitochondria-specific fluorescent dye MitoTracker CMXRos (Thermo Fisher Scientific, M7514) was used to monitor mitochondrial morphology according to the manufacturer’s instructions. Briefly, cells were seeded on coverslips coated with Poly-D-lysine (Sigma-Aldrich, P6407) and treated with fresh growth media containing 100ng/ml MitoTracker CMXRos (Invitrogen, M7512) at 37°C for 15-30 minutes. Cells then were washed twice with growth media and fixed with pre-warmed media containing 3.7% formaldehyde solution for 15 minutes at 37°C. After fixation, cells were washed with PBS three times, treated with 0.2% of Triton X-100 for 10 minutes at room temperature, then treated with 300nM of DAPI in PBS (Invitrogen, D3571). Coverslips were mounted on glass slides with 25μl of Fluoromount-G™ Mounting Medium (Invitrogen, 00-4958-02). Fluorescence imaging was carried out on DeltaVision high resolution fluorescent microscope (GE Healthcare) equipped with SoftWork software. Raw images were processed using ImageJ. Mitochondria shape and number were analyzed by the MAT-LAB based macro, MicroP according to the author’s instructions59 (link). Up to 10 images at 60x magnification per sample were analyzed.
Mitotracker cmxros
Manufactured by Thermo Fisher Scientific
Sourced in United States
MitoTracker CMXRos is a fluorescent dye that selectively labels mitochondria in live cells. It is a cell-permeant dye that accumulates in active mitochondria due to their negative membrane potential.
Automatically generated - may contain errors
Lab products found in correlation
Axio imager m2 microscope, by Zeiss (3 mentions)
Volocity software, by PerkinElmer (3 mentions)
Lo flo, by Formedium (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Fl 145, by Santa Cruz Biotechnology (3 mentions)
Mitotracker cmxros, by FlowJo (3 mentions)
Lsm 780 confocal microscope, by Zeiss (2 mentions)
Lab tek 2, by Thermo Fisher Scientific (2 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (2 mentions)
Facsverse flow cytometer, by BD (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Sp5 confocal microscope, by Leica (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Lysotracker dnd 99, by Thermo Fisher Scientific (2 mentions)
Blockaid, by Thermo Fisher Scientific (2 mentions)
Tcs sp5 x confocal microscope, by Leica (2 mentions)
80 protocols using mitotracker cmxros
1
Mitochondrial Morphology Analysis
2
Analyzing Mitochondrial PRDX3 Localization
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Caco-2 cells treated with 200 nM MitoTracker CMXRos (Invitrogen) were cultured in a glass-bottomed vessel for 30 min at 37 °C protected from light, washed with PBS, fixed in 4% PFA for 30 min at room temperature, and treated with 0.1% Triton X-100 in PBS for 10 min for permeation. The cells were then washed with PBS, blocked with 1% BSA for 1 h at 37 °C, and incubated with anti-PRDX3 antibody overnight at 4 °C. The cells were then washed again, incubated with Alexa Fluor 488-conjugated secondary antibody (Proteintech) for 1 h at room temperature, and washed with PBS. The nuclei were stained using a DAPI solution for 5 min at room temperature, and the cells were subsequently visualized under a laser confocal microscope (Leica TCS SP5II, Germany).
3
Immunofluorescence Imaging of Cell Morphology
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Cells seeded on glass coverslips were fixed with 4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature, rinsed with 10% FBS/PBS, and incubated for 1 h with primary antibodies in 10% FBS/PBS supplemented with 0.2% saponin. After washing three times with 10% FBS/PBS to remove unbound antibody, the cells were incubated for 1 hour with the appropriate fluorescently-conjugated secondary antibodies diluted in 10% FBS/PBS containing 0.2% saponin. Coverslips were washed three times with 10% FBS/PBS, once with PBS, and then mounted on a slide.
To visualize morphology of the cell architecture and mitochondrial network, the cells were incubated with 200 nM MitoTracker CMXRos (Invitrogen, San Diego, CA, USA) in the dark at 37 °C for 10 min. The cells were then briefly rinsed with the medium under the same conditions and fixed. CMXRos is a cationic lipophilic dye, preferentially sequestered into mitochondria and reacting with thiols of proteins in the mitochondrial matrix. Actin filaments were stained by Alexa Fluor 488 phalloidin (Invitrogen, Eugene, OR, USA).
Confocal fluorescence images and image stacks (0.3 μm) were collected using a Zeiss LSM780 confocal microscope with a 63× oil immersion objective. Images were acquired from randomly selected fields of nonconfluent cells. All images were processed and analyzed using Zeiss LSM780 software.
To visualize morphology of the cell architecture and mitochondrial network, the cells were incubated with 200 nM MitoTracker CMXRos (Invitrogen, San Diego, CA, USA) in the dark at 37 °C for 10 min. The cells were then briefly rinsed with the medium under the same conditions and fixed. CMXRos is a cationic lipophilic dye, preferentially sequestered into mitochondria and reacting with thiols of proteins in the mitochondrial matrix. Actin filaments were stained by Alexa Fluor 488 phalloidin (Invitrogen, Eugene, OR, USA).
Confocal fluorescence images and image stacks (0.3 μm) were collected using a Zeiss LSM780 confocal microscope with a 63× oil immersion objective. Images were acquired from randomly selected fields of nonconfluent cells. All images were processed and analyzed using Zeiss LSM780 software.
4
Molecular Characterization of Viral Proteins
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ITZ was purchased from Santa Cruz Biotechnology and 25-Hydroxycholesterol (25-HC) from Sigma Aldrich (St. Louis, MO, USA). Rabbit polyclonal anti-OSBP (Sigma) was used at 1:50 dilution, anti-PI4Kβ (Millipore, Burlington, MA, USA) at 1:300 dilution, anti-ACBD3 (Sigma-Aldrich) at 1:100 dilution and mouse monoclonal antibody to PDI (ab2792 abcam) at 1:1000; MVB were labeled with anti-CD63 antibody (Clone H5C6; Developmental Studies Hybridoma Bank, University of Iowa) at 1:200 dilution. MitoTracker CMXRos was obtained from Invitrogen (Waltham, MA, USA) and used at 100 nM. Other primary antibodies used were monoclonal antibodies against the virus major capsid protein p72 (Ingenasa, Madrid, Spain) used at a working dilution of 1:1000, anti-p30 antibody at 1:100 dilution (kindly given by Dr. J.M. Escribano, INIA) and swine anti-p54 antibody at 1:100. As secondary antibodies, we used anti-mouse immunoglobulin G (IgG) antibody conjugated to Alexa Fluor 594 and anti-rabbit IgG antibody conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA), both at 1:200 dilutions and FITC-conjugated anti-mouse inmmunoglubulins 1:50, diluted in FACS buffer (Dako, Agilent, Santa Clara, CA, USA).
5
Mitochondrial Staining and Imaging of Infected RBCs
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For mitochondrial staining, infected RBCs were stained with 10 nM MitoTracker CMXRos (Invitrogen; M7512) for 30 min at 37°C. Cells immobilised in PHA‐E‐coated coverslips were then fixed with 2% (v/v) paraformaldehyde/ 0.008% (v/v) glutaraldehyde for 15 min, washed with PBS followed by permeabilisation with 0.1% TX‐100 in PBS for 10 min and washed (adapted from (Tonkin etal, 2004 )). Cells were probed with rat anti‐HA (Roche; 3F10) and mouse anti‐BiP (Bridgford etal, 2018 ) at 1:1,000 in 3% BSA/PBS. Secondary antibodies used were anti‐rat Alexa 647 (Invitrogen; A21247) and anti‐mouse Alexa 488 (Invitrogen; A11029) at 1:300 in 3% BSA/PBS. Nuclear staining was performed with 2 µg/ml DAPI and washed prior to mounting. Images were taken using the DeltaVisionElite (GE Lifesciences) and processed using ImageJ.
6
Rho0 Cell Generation via Ethidium Bromide
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HDFn cells were incubated in DMEM containing 10% FBS (Sigma-Aldrich), 1 mmol/L pyruvate, 50 mg/L uridine, and 100 g/L ethidium bromide to generate Rho0 cells. Cells were cultured in a humidified 5% CO2 incubator at 37 °C and the medium was changed every 2 days. Cells were cultured for 30–35 days and the generation of Rho0 cells was confirmed by PCR and RT-PCR using The Human Mitochondrial DNA (mtDNA) Monitoring Primer Set (Cat. #7246, Takara Bio Inc.) and data analysis was performed according to the manufacturer’s instructions. To visualize mitochondrial network in HDFn and Rho0 cells, cells were stained with MitoTracker CMXRos (Invitrogen) according to the manufacturer’s instructions.
7
Fluorescent Parasite Visualization
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For sample preparation, 100μL of parasite culture was obtained. Parasites were incubated with 30nM MitoTracker CMX-Ros (Invitrogen) and 1μg/mL 4′, 6-diamidino-2-phenylindole (DAPI) for 30 minutes at 37°C. Cells were then washed three times with 100μL of CMA media and incubated for 5 minutes at 37°C after each wash. Cells were resuspended in 20μL of CMA and then pipetted onto slides and sealed with wax for observation on a Zeiss AxioImager M2 microscope. A series of images spanning 5μm were acquired with 0.2μm spacing and images were deconvolved using VOLOCITY software (PerkinElmer) to report a single image in the z-plane.
8
Mitochondrial Imaging of Live Cells
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Log phase cells were prepared for imaging by washing 1 time and resuspending in Lo-Flo (Formedium, Hunstanton, Norfolk, UK). Cells were stained with 0.1 µM final concentration of Mitotracker CMXRos, (Invitrogen, Thermo Fisher Scientific, Grand Island, NY, USA) washed 3 times with Lo-Flo and placed in Nunc Lab-TekII 4-well chambered coverglass for imaging. To image the treated cells, we utilized a Zeiss laser scanning LSM 880 confocal microscope (Zeiss, Stockholm, Sweden) with a pinhole setting of 144 µm. This allows for a 1.1 µm optical slice. Cells were imaged on a minimum of 3 separate occasions with approximately 5 time-lapse images obtained each time.
9
Mitochondrial Dynamics in Live Cells
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NSC34 cells were plated on poly-D-lysine/fibronectin coated 35 mm glass-bottomed petri dishes (14 mm microwell diameter, glass thickness No. 1.5) (MatTEK). Mitochondria were labeled with 25 nM MitoTracker CMXRos (Invitrogen) for 30 min in pre-warmed DMEM, followed by three five minute washes with DMEM. Two channel, single plane imaging of live-cells was performed in a heated chamber at 37 °C with 5% CO2 (Okolab) using a 60X objective lens (Plan ApoVC NA 1.4 oil) at an acquisition rate of 0.25 fps.
10
MitoTracker Imaging in Live Cells
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MitoTracker staining was performed as described previously, with some modifications63 (link). MitoTracker CMXRos (Invitrogen) was dissolved in dimethylsulfoxide (DMSO) to a stock concentration of 10 mM and was added to animals on the plates to a final concentration of 10 μM for 6 hours before analyses. The images were photographed by using the Nikon A1si/Ni-E upright confocal microscope with 100×, 1.4 NA oil-immersion objective lens. Confocal microscopy data were acquired at the Brain Research Core Facilities in Korea Brain Research Institute (KBRI).
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