Immunofluorescence was performed in cultured BAs and the Cos-7 cell line. Briefly, samples were fixed in 4% PFA for 10 min, quenched with 100 nM glycine for 10 min, and permeabilized and blocked in blocking buffer (PBS containing 5% goat serum, 2% bovine serum albumin (BSA), 0.2% Triton X-100, and 0.1% sodium azide) for 1 h at RT. Samples were subsequently incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. After washing with PBS, samples were incubated with secondary antibodies and DAPI (Invitrogen, cat#D1306) for 1 h at RT. Mature BAs were stained with Bodipy 493/503 (Invitrogen, cat#D3922) and DAPI. For MitoTracker (Cell Signaling Technology, cat#9082) staining, live cells were incubated with FBS-free DMEM containing 0.2% MitoTracker for 50 min, then fixed with iced-cold methanol for 20 min. Antibodies used for immunofluorescence staining are listed in Supplementary Table 1 . Primary antibodies and dilutions were used as follows: C18orf19 (FAM210A, Invitrogen, PA5-53146; 1:300), c-Myc (Santa Cruz Biotechnology, sc-40; 1:300), Tom20 (Santa Cruz Biotechnology, sc-11415; 1:300). Fluorescent images were captured using a Leica DM6000B microscope.
Mitotracker
Manufactured by Cell Signaling Technology
Sourced in United States
MitoTracker is a fluorescent stain that can be used to visualize and track mitochondria in live cells. It is a cell-permeant dye that accumulates in active mitochondria, allowing for the observation of mitochondrial morphology and distribution within the cell.
Automatically generated - may contain errors
Lab products found in correlation
Live cell imaging solution, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (2 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (2 mentions)
Te2000 u microscope, by Nikon (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
A1rs confocal microscope, by Nikon (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Rnase a, by New England Biolabs (1 mentions)
Eclipse ti u fluorescent microscope, by Nikon (1 mentions)
Facscanto cytometer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Iris digital cell imaging system, by Logos Biosystems (1 mentions)
Mito sox, by Logos Biosystems (1 mentions)
Tom20, by Santa Cruz Biotechnology (1 mentions)
Dm6000b microscope, by Leica (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Tetramisole, by Merck Group (1 mentions)
Homocysteine, by Merck Group (1 mentions)
16 protocols using mitotracker
1
Immunofluorescence Staining of Cultured Cells
2
Monitoring UPR Activation in C. elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine UPRmt or UPRER activation, strains carrying hsp‐6p::GFP or hsp‐4p::GFP were utilized. Synchronized worms were visualized at of day 1 of adulthood. For the fluorescence imaging, worms were anesthetized with M9 buffer containing 20 mM sodium azide, and an Olympus BX63 microscope was used. Worm GFP intensity was then quantified with ImageJ software. In SAM, methionine, and homocysteine supplementation experiments, P0 worms were grown on empty vector or RNAi bacteria from hatching. F1 eggs were then transferred on plates seeded with UV‐killed bacteria and supplemented with the vehicle, 2 mM SAM (Sigma‐Aldrich, A2408), 2 mM L‐methionine (Sigma‐Aldrich, M5308), and 2 mM homocysteine (Sigma‐Aldrich, 69453). Day 1 adult F1 worms were then scored for GFP intensity.
For monitoring the subcellular localization of SLC‐25A26::GFP or TRMT‐10C.2::GFP, worms were mounted on 2% agarose pads with 50 mM tetramisole (Sigma, T1512). Images were acquired using Zeiss Elyra 7 microscope and Zeiss LSM 900 laser scanning confocal microscope, respectively.
For Mitotracker Red staining, L4 worms carrying TRMT‐10C.2::GFP were cultured on UV‐killed bacteria with 2 μM Mitotracker (Cell Signaling Technology, 8778) for 24 h. Worms were subsequently transferred to stain‐free plates for 30 min to eliminate the excess stain in the gut lumen before proceeding with image analysis.
For monitoring the subcellular localization of SLC‐25A26::GFP or TRMT‐10C.2::GFP, worms were mounted on 2% agarose pads with 50 mM tetramisole (Sigma, T1512). Images were acquired using Zeiss Elyra 7 microscope and Zeiss LSM 900 laser scanning confocal microscope, respectively.
For Mitotracker Red staining, L4 worms carrying TRMT‐10C.2::GFP were cultured on UV‐killed bacteria with 2 μM Mitotracker (Cell Signaling Technology, 8778) for 24 h. Worms were subsequently transferred to stain‐free plates for 30 min to eliminate the excess stain in the gut lumen before proceeding with image analysis.
3
Lysosome and Mitochondria Imaging in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells labeled with LysoTracker (Invitrogen, L7528) and MitoTracker (Cell Signaling Technology, 8778) were transferred to a microscope stage and maintained at 37°C. Pseudo-TIRF microscopy (oblique illumination) experiments were performed using a X100 1.45 numerical aperture TIRF objective (Nikon) on a Nikon TE2000U microscope custom-modified with a TIRF illumination module as described [11 (link)]. Images were acquired on a 14-bit cooled charge-coupled device camera (Hamamatsu) controlled through NIS-Elements software. For live cell imaging, the images were recorded using 200–500-ms exposures depending on the fluorescence intensity of the sample. The track speed mean was then analyzed using Imaris as described before [11 (link),39 (link)]. Wild-type and ctns-/- cells were analyzed comparatively by maintaining exposure time and gain throughout the experiment. Length of mitochondria were analyzed using the “Measure” tool in ImageJ software.
4
Immunofluorescence Staining of Cell Lines
5
Imaging Mitochondrial Dynamics in MEFs
6
Visualizing Mitochondria and Lipids in 3T3-L1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3T3-L1 cells were grown as described in ‘3T3-L1 culture, differentiation and treatment’. At day 7 post induction cells were incubated in a medium containing 50 μM of Mitotracker (Cell Signaling, 9082P) for 30 min. Cells were then washed with PBS and fixed with 4% formaldehyde during 10 min and rinsed 3 times with PBS. Lipid droplets were stained with a Bodipy 493/503 (D3922, Thermo Fisher Scientific) in a staining solution (1 μg/ml Bodipy 493/503, 150 mM NaCl) for 10 min at room temperature. Nuclei were stained with DAPI (0.5 μg/ml).
7
Visualizing Nucleic Acids in C. elegans and Mammalian Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For C. elegans, animals, pre-stained with EtBr (8 µg/ml or 20.3 µM) and SYBR gold (1/5000) followed by hypoxia, were gonad dissected, fixed (4% formaldehyde, Electron Microscopy Sciences), permeabilized (PBS + 0.1% triton-X 100, Sigma), and treated with DNase I (50 U/ml, New England BioLabs) or RNase A (500 mg/ml, New England BioLabs). The gonads were then mounted and imaged.
For mammalian cells, the H9c2, embryonal rat heart-derived cells were obtained from the ATCC (atcc.org) and were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco) supplemented with 10% fetal calf serum (Gibco). The cells grown on glass cover slips, with less than 80% confluence, were prestained with EtBr (1.58 µg/ml or 4 µM) and SYBR gold (1/10,000X) or MitoTracker (0.25 µM) (Cell Signaling) followed by sodium azide (NaN3, 100 mM, 3 h, Sigma) treatment. For MitoTracker/EtBr staining, cells were immediately mounted and imaged by confocal microscopy. For DNase and RNAse experiments, cells were fixed (4% formaldehyde, Electron Microscopy Sciences), permeabilized (PBS + 0.1% triton-X 100, Sigma), and treated with DNase I (50 U/ml, New England BioLabs) or RNase A (500 mg/ml, New England BioLabs). The cells were then mounted and imaged.
For mammalian cells, the H9c2, embryonal rat heart-derived cells were obtained from the ATCC (atcc.org) and were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco) supplemented with 10% fetal calf serum (Gibco). The cells grown on glass cover slips, with less than 80% confluence, were prestained with EtBr (1.58 µg/ml or 4 µM) and SYBR gold (1/10,000X) or MitoTracker (0.25 µM) (Cell Signaling) followed by sodium azide (NaN3, 100 mM, 3 h, Sigma) treatment. For MitoTracker/EtBr staining, cells were immediately mounted and imaged by confocal microscopy. For DNase and RNAse experiments, cells were fixed (4% formaldehyde, Electron Microscopy Sciences), permeabilized (PBS + 0.1% triton-X 100, Sigma), and treated with DNase I (50 U/ml, New England BioLabs) or RNase A (500 mg/ml, New England BioLabs). The cells were then mounted and imaged.
8
Quantifying Cell Fusion Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After cell differentiation, cells were stained with 50 nM MitoTracker (Cell Signaling Technology) for 45 min at 37 °C. Cells were then fixed with cold methanol for 10 min, followed by washing 3× with PBS for 5 min. Cells were permeabilized with 0.05% Triton X-100 solution, blocked with 1% bovine serum albumin solution for 30 min, and incubated overnight at 4 °C with the primary antibody. Cells were then washed 3× with PBS for 5 min and incubated with a goat anti-mouse immunoglobulin G, Flamma® 488-labeled secondary antibody (Invitrogen, Waltham, MA, USA) for 1 h in the dark. Nuclei were stained with 4′,6-diamidino-2-phenylindole and observed using an Eclipse Ti-U fluorescent microscope (Nikon, Tokyo, Japan). The fusion index was determined by calculating the ratio of nuclei present in MHC-positive cells with 2 or more nuclei among the total nuclei in the randomly selected region.
9
Quantifying Mitochondrial Fragmentation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The morphology of mitochondria was examined by MitoTracker staining (Cell Signaling, 9074) as previously described [22 (link)]. The percentage of mitochondrial fragmentation was calculated by comparing the number of cells with fragmented mitochondria to the total number of cells.
10
Multiparameter Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For FACS analysis, 1 × 106 cells were used per sample. Cells were blocked for 15 min in PBS, 2% BSA on ice in a total volume of 100 μL. The staining step was carried out on ice for one hour with the appropriate antibody or incubated in the corresponding isotype control diluted as suggested by the manufacturer’s protocol. The surface expression of RANK (R12-31) IgG2a (RTK2758), CD11b (M1/70), CD80 (16-10A1), CD86 (GL-1), F4/80 (BM8) was quantified by flow cytometry by using FACS Canto cytometer (BD Biosciences, Heidelberg, Germany) and BD FACS Diva Software. For the live/dead staining, cells were washed once with DPBS and were stained with SYTOX dye. The cells could be analysed within the FITC channel directly, without further incubation or washing. For measuring mitochondrial activity, the cells were stained with 100 nM MitoTracker (Cell Signaling Technology, Leiden, The Netherlands). After 30 min incubation, the cells were washed with PBS and analysed within the APC-Cy7 channel.
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!