For determination of localization of mitochondrial stress proteins in chondrocytes, a modified Mito-Tracker Red CMXRos kit (Beyotime, China) was used according to manufacturer’s instructions to label mitochondria, at an excitation of 579 nm and an emission of 599 nm. Cultured cells were washed thrice with PBS and fixed in 4% paraformaldehyde for 20 min. Cells were first treated with CMXRos for 20 min. The cells were permeabilized for 10 min with 0.1% Triton X-100 and blocked with 5% BSA for 30 min at room temperature. Next, the cells were stained with anti-HSP10, anti-ClpP, or anti-LONP1 antibodies (1:500; Bioss, Beijing, China) with 1% BSA overnight at 4 °C. The next day, the cells were washed three times with PBS for 5 min, incubated with the corresponding fluorescent-labeled secondary antibody (FITC, 1:200 Boster, Wuhan) for 1 h in the dark, washed three times with PBS for 5 min, and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Images were captured using a confocal microscope (Zeiss, Germany). FITC was analyzed with excitation at 495 nm and emission at 591 nm. DAPI was analyzed with excitation at 353 nm and emission at 465 nm.
Mito tracker red cmxros kit
Manufactured by Beyotime
Sourced in China
The Mito-Tracker Red CMXRos kit is a fluorescent dye used to stain and visualize mitochondria in living cells. It is a cell-permeant, red-fluorescent dye that specifically labels active mitochondria.
Automatically generated - may contain errors
Lab products found in correlation
Fv1000 confocal microscope, by Olympus (2 mentions)
Confocal microscope, by Zeiss (1 mentions)
Fluorescein isothiocyanate (fitc), by Boster Bio (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Cck 8 kit, by Beyotime (1 mentions)
Multiskan fc, by Thermo Fisher Scientific (1 mentions)
Fv3000, by Olympus (1 mentions)
Mitosox green reagent, by Thermo Fisher Scientific (1 mentions)
Atp content assay kit, by Solarbio (1 mentions)
9 protocols using mito tracker red cmxros kit
1
Mitochondrial Stress Protein Localization in Chondrocytes
2
Colocalization of ALR and ACSL4 in Mitochondria
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To determine whether ALR and ACSL4 colocalize in mitochondria, the MitoTracker Red CMXRos Kit (Beyotime Biotech Co., Ltd.) was used to label viable mitochondria. The HK‐2 cells were seeded in confocal microscopy dishes and allowed to adhere to the substrate overnight. After H/R injury, the cells were washed with phosphate‐buffered saline three times and stained with trimethylrhodamine, methyl ester, for 20 min, followed by fixation with 4% paraformaldehyde. The cells were washed, permeabilized, blocked with 0.5% BSA and incubated overnight at 4°C with primary antibodies (ACSL4, 1:100 or ALR, 1:50). On the following day, the cells were incubated with a fluorescein isothiocyanate (FITC)‐ or a tetramethylrhodamine isothiocyanate (TRITC)‐conjugated secondary antibody for 60 min at room temperature in the dark. The cell nuclei were counterstained with DAPI. The cells were observed under a confocal laser‐scanning microscope (Nikon A1R + Confocal Microscope, Nikon, Japan).
3
Apoptosis Detection in U251 Cells
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A MitoTracker red CMX Ros kit (Beyotime Biotechnology, Shanghai, China) was used to identify apoptotic U251 cells treated with 25, 50 or 100 nM bufalin for 24 h. An Olympus FV1000 confocal microscope was used to detect red fluorescence at λex/λem = 579/599 nm, green fluorescence at λex/λem λex/λem = 492/520 nm, and blue fluorescence at λex/λem = 350/461 nm.
4
Evaluating Ochratoxin A Cytotoxicity in HepG2 Cells
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Human hepatoma HepG2 cells were cultured in Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum at 37 °C in an incubator with 5% CO2 and 95% air. The OTA standard was dissolved in DMSO. HepG2 cells were treated with different concentrations of OTA, and the IC50 value was determined. The optimal concentration was selected for subsequent experiments, and the control group was exposed to DMSO only. The cells were treated with OTA, degradation products, and zymotic fluid to detect various cell parameters. Cell viability was assessed with the CCK-8 kit (Beyotime Biotechnology, Shanghai, China). The cell viability (CV) was calculated as follows:
AS was the experimental group, AC was the control group, and AB was the blank group.
Cell morphology was observed under an inverted microscope. Reactive oxygen species (ROS) activity and mitochondrial membrane potential were assessed with the ROS Assay kit and the Mito-Tracker Red CMXRos kit (Beyotime Biotechnology, Shanghai, China).
AS was the experimental group, AC was the control group, and AB was the blank group.
Cell morphology was observed under an inverted microscope. Reactive oxygen species (ROS) activity and mitochondrial membrane potential were assessed with the ROS Assay kit and the Mito-Tracker Red CMXRos kit (Beyotime Biotechnology, Shanghai, China).
5
Mitochondrial Function and Oxidative Stress Assay
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MMP detection was performed by Mito-Tracker Red CMXRos kit (Beyotime, C1049B). After treatment with PS-MPs for 24 h, the cells were incubated with Mito-Tracker Red CMXRos (50 nM) for 20 min at 37°C. The cells were then washed thrice with PBS before the detection of red fluorescence intensity by CLSM (Olympus FV3000) and quantified by ImageJ software.
ATP Content Assay Kit (Solarbio, BC0300) was used for ATP content assay. Briefly, the cell were lysed and centrifuged at 12,000 r/min at 4°C for 5 min, and supernatant were then collected. The ATP content was determined according the manufacture’s instruction. The samples were measured by a microplate reader (Thermo Scientific, Multiskan FC).
The detection of mtROS was performed by MitoSOX™ Green reagent (Thermo Fisher, M36008). TM4 cells were seeded in 6-well plate and exposed to PS-MPs for 24 h. Followed by washing with PBS, the cells were incubated with 10 µM MitoSOX™ for 20 min at 37°C. The fluorescent images were visualized by CLSM (Olympus FV3000) and quantitative analysis by ImageJ software.
ATP Content Assay Kit (Solarbio, BC0300) was used for ATP content assay. Briefly, the cell were lysed and centrifuged at 12,000 r/min at 4°C for 5 min, and supernatant were then collected. The ATP content was determined according the manufacture’s instruction. The samples were measured by a microplate reader (Thermo Scientific, Multiskan FC).
The detection of mtROS was performed by MitoSOX™ Green reagent (Thermo Fisher, M36008). TM4 cells were seeded in 6-well plate and exposed to PS-MPs for 24 h. Followed by washing with PBS, the cells were incubated with 10 µM MitoSOX™ for 20 min at 37°C. The fluorescent images were visualized by CLSM (Olympus FV3000) and quantitative analysis by ImageJ software.
6
MitoTracker Red CMXRos Staining Protocol
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A MitoTracker Red CMXRos kit was purchased from Beyotime Biotechnology Co., Ltd. (C1049B, Wuhan, China) in order to carry out MitoTracker staining. After stimulating bMECs with 5 ng/mL TGF-β1 for 48 h. (1) Preparation of storage: 50 μL Mito-Tracker Red CMXROS solution was added to 420 μL of water-free anhydrous dimethyl sulfoxide (DMSO). After mixing, the storage solution with a concentration of 200 μm was obtained, and stored at −20 °C. (2) Preparation of the working solution: A small amount of 200 μm storage solution was added to the cell culture solution at a ratio of 1:1000, so that the final concentration was 20 nm, and the working liquid was heated to 37 °C before use. (3) When the cells were cultivated to a certain density in the cell culture plate or a Petri dish, the cell culture medium was removed, the prepared work solution was added, and incubated for 15 min at 37 °C. (4) The working liquid was removed and fresh cell culture liquid was added with pretemperature cultivation at 37 °C. (5) The results were observed or detected with a fluorescent microscope.
7
Mitochondrial Function Assessment of AFCs
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MitoTracker Red CMXRos kit (C1049B, Beyotime) was used to detect the mitochondrial function of AFCs. Working solution was configured according to the reagent manufacturer’s instructions. AFCs were incubated with working solution at 37 °C for 30 min in the dark. Nuclei were stained with Hoechst 33,258. The samples were observed under the inverted fluorescence microscope.
8
Bufalin-Induced Apoptosis in U251 Cells
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The Mitotracker red CMX Ros kit (Beyotime Biotechnology, Shanghai, China) was used to probe apoptosis of U251 cells treated with 25, 50 and 100 nM bufalin for 24 h. Use Olympus FV1000 confocal microscope to detect red uorescence at λ ex / λ em = 579/599 nm, green uorescence at λ ex / λ em = 492/520 nm, and blue uorescence at λ ex / λ em = 350/461 nm.
9
Mitochondrial Membrane Potential in Bladder Cancer
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Mitophagy is accompanied by the attenuation of mitochondrial membrane potential. The effect of CA on the mitochondrial membrane potential of bladder cancer cells was detected by Mito-Tracker Red CMXRos kit (Beyotime, China, C1049B) according to the manufacturer's instructions.
Xenograft in nude mice 10 six-week-old BALB/C-nude mice were injected subcutaneous with 10 7 bladder cancer SW780 cells.
When the tumor grew to 100mm 3 , the mice were randomly divided into two groups with 5 mice in each group. The CA-treatment group was intraperitoneally injected once every 2 days at a dose of 8 mg/kg (CA was rst dissolved in DMSO and then dissolved in corn oil), while the control group was given DMSO and corn oil at the same volume at the same IP injection frequency. After administration, the tumor volume was measured every 2 days and the mice were weighed every 3 days. After 10 doses injection, the mice were sacri ced, and the subcutaneous tumors were taken, photographed and weighed, then xed with 10% formalin, dehydrated and made into para n blocks for subsequent studies. At the time of sacri cing mice, blood was collected for routine blood measurement.
Xenograft in nude mice 10 six-week-old BALB/C-nude mice were injected subcutaneous with 10 7 bladder cancer SW780 cells.
When the tumor grew to 100mm 3 , the mice were randomly divided into two groups with 5 mice in each group. The CA-treatment group was intraperitoneally injected once every 2 days at a dose of 8 mg/kg (CA was rst dissolved in DMSO and then dissolved in corn oil), while the control group was given DMSO and corn oil at the same volume at the same IP injection frequency. After administration, the tumor volume was measured every 2 days and the mice were weighed every 3 days. After 10 doses injection, the mice were sacri ced, and the subcutaneous tumors were taken, photographed and weighed, then xed with 10% formalin, dehydrated and made into para n blocks for subsequent studies. At the time of sacri cing mice, blood was collected for routine blood measurement.
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