To analyze intracellular reactive oxygen species (ROS) levels in hepatocytes (HepG2 cells and L02 cells), the cells were exposed to a 10 μM DCFH-DA probe at 37 °C for 20 to 30 min. After that, the cells were washed twice with PBS to eliminate any unbound fluorescent probe. The ROS were visualized as green fluorescence and captured using a fluorescence microscope (Thermo Fisher Scientific). To determine the production of mitochondrial ROS in hepatocytes, the cells were treated with 2.5 μM MitoSOX Red probe for 20 to 30 min. Subsequently, the cells were washed twice with PBS to remove any excess fluorescent probe. The mitochondrial ROS were visualized as red fluorescence and captured using a fluorescence microscope (Thermo Fisher Scientific).
Fluorescence microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, China
A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence to study the properties of organic or inorganic substances. It is designed to detect and analyze the emission of light from a sample that has been excited by a specific wavelength of light.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (4 mentions)
Image pro plus 6, by Media Cybernetics (3 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (3 mentions)
Microplate reader, by Bio-Rad (2 mentions)
5 ethynyl 20 deoxyuridine edu assay kit, by RiboBio (2 mentions)
Rabbit anti insulin antibody, by Abcam (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Reactive oxygen species assay kit, by Beyotime (2 mentions)
Calcein acetoxymethyl ester, by Thermo Fisher Scientific (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Edu reagent, by Solarbio (1 mentions)
Hoechst, by RiboBio (1 mentions)
Click itr edu kit, by RiboBio (1 mentions)
M199 medium, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Mitotracker green, by Cell Signaling Technology (1 mentions)
108 protocols using fluorescence microscope
1
Intracellular ROS Quantification in Hepatocytes
2
Cell Proliferation Assay with EdU Staining
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Cells seeded in 96-well plates were incubated with 100 μL EdU reagent (Solarbio) for 2 h. The cells were washed with phosphate-buffered saline (PBS) and stained with Apollo reagent. Next, the cells were stained with methanol, fixed, permeated, and then cultured with 100 μL 4′,6-diamidino-2-phenylindole (DAPI) reaction solution (Sigma–Aldrich, Merck KGaA, Darmstadt, Germany). Positive EdU labeling, namely, the viability of cells, was determined under a fluorescence microscope (Thermo Fisher Scientific).
3
Cell Proliferation Quantification using EdU Assay
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The 5-ethynyl-2′-deoxyuridine (EdU) assay was performed using the Click-iTR EdU Kit (RiboBio, China) according to the manufacturer’s instructions. Specifically, cells were plated in 96-well plates and treated after adhesion. A total of 100 μL of culture medium containing 50 mM EdU was added to each well, and, 4 h later, the cells were fixed with 4% formaldehyde for 30 min. After washing, the cells were incubated with a solution in the kit for 30 min and stained with Hoechst (RiboBio, China) to identify nuclei, and images were captured with a fluorescence microscope (Thermo Fisher Scientific, USA). EdU-positive cells were determined using ImageJ 1.50i software (https://imagej.en.softonic.com/ ).
4
Biocompatibility Assessment of Hydrogels
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L929 fibroblasts were cultivated for the biocompatibility test of AAm-AAc/CNC, AAm-AAc-Fe3+/CNC, and AAm-AAc-Fe2+/CNC hydrogels. Samples of these hydrogels were made and freeze-dried in advance. They were later soaked in cell culture medium and disinfected by ultraviolet irradiation for 24 h. The leachates and fibroblasts were seeded in a 24-well culture plate and co-cultured for 24 h and 48 h respectively. Cell viability was observed under a fluorescence microscope (Thermo Fisher Scientific Co., Ltd, USA) after stained by calcein AM and propidium iodide (PI).
5
Cell Proliferation Assay Using EdU Staining
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Cells were seeded in a 96-well culture plate at a density of 1 × 104 cells/well in 100 μL of culture medium. Then, 100 μL 5-ethynyl-2´-deoxyuridine (EdU) medium (50 μmol, Ribobio, Guangzhou, Guangdong, China) was added to each well and the plate was incubated at 32°C for 2 h. DNA and EdU staining solution were subsequently added to each well to identify living cells (blue) and proliferating cells (red) based on the manufacturer’s protocol, respectively. Finally, the cells were observed under a fluorescence microscope (ThermoFisher).
6
EdU Assay for Cell Proliferation
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The EdU Kit (RiboBio, Guangzhou, China) was used to perform the 5-ethynyl-2′-deoxyuridine (EdU) assay according to the manufacturer’s instructions. Cells were planted in 96-well plates and then treated after adhesion. Each well received a total of 100 μL of culture medium containing 50 mM EdU. Cells were fixed with 4% formaldehyde for 30 min. After washing, the cells were incubated for 30 min with a solution from the kit before being stained with Hoechst to identify nuclei. A fluorescence microscope was used to capture the images (Thermo Fisher Scientific, Waltham, MA, USA). Image J was used to identify EdU-positive cells.
7
Photodynamic Therapy for ROS Generation
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HNE-1 cells were plated into 24-well plates at 1.4 × 105 cells per well and incubated with 1 ml of complete medium. Agents specific for each group were added to the plate at a concentration of 5 μg/ml and cultured for 6 h. For the PDT test, 1 mM fluorescent dye (DCFH-DA) was added to each well, with NIR irradiation of 0.98 W/cm2 for 1 min being applied to the corresponding wells. As the control sample, PBS was irradiated with the same irradiation parameters as above. After continued culture for 2 h, the production of ROS was observed under a fluorescence microscope (Thermo, America) (Figure 4B ). We used ImageJ software for general quantitative analysis.
8
Quantifying THP-1 Cell Adhesion to HUVECs
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THP-1 cells were labeled with 5 µmol/L calcein-acetoxymethyl ester (Invitrogen) and cultured at 37 °C in a 5% CO2 incubator at 90% humidity for 30 min. The labeled THP-1 cells were washed twice with PBS (Invitrogen) and resuspended in M199 medium (Invitrogen). The HUVEC medium was removed and the suspended THP-1 cells (HUVECs: THP-1 cells =30:1) were added to the confluent monolayer. Then, the plates were put back into the incubator. After 3 h, the supernatant was removed, and the plates were washed with PBS and then carefully dried with filter paper. The adherent calcein-acetoxymethyl ester labeled THP-1 cells were counted using a fluorescence microscope (Thermo Fisher Scientific). Each dish was divided into eight quadrants. The middle position (200× visual field) of each quadrant was selected for counting, and the average was taken as the number of adherent THP-1 cells from each quadrant.
9
Imaging Transplanted Mitochondria via Fluorescent Probes
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For in vitro imaging,
MitoTracker Green and Red (Cell Signaling Technology, Danvers, MA,
USA) were used to incubate with isolated mitochondria and receptor
cells, respectively. The combination of Cy5 marked PEP–TPP
and MitoTracker Green-labeled mitochondria was imaged by a confocal
microscope (Thermo Fisher Scientific). To observe the dynamic internalization
of transplanted mitochondria, PEP(Cy5)–TPP–mitochondria
was added into the culture medium. Then the fluorescence intensity
of MitoTracker Green and Cy5 was detected by the Lionheart FX living
cell imaging analysis system (BioTek, Winooski, VT, USA).
For
in vivo imaging, to detect the retention of transplanted PEP(Cy5)–TPP–mitochondria
(GFP), mouse hearts were imaged by a fluorescence detection system
(IVIS Lumina XRMS, USA) after reperfusion for 3, 6, and 24 h. Besides,
the Cox4i1-GFP marked mitochondria of formalin-fixed heart tissue
sections were imaged with a fluorescence microscope (Thermo Fisher
Scientific).
MitoTracker Green and Red (Cell Signaling Technology, Danvers, MA,
USA) were used to incubate with isolated mitochondria and receptor
cells, respectively. The combination of Cy5 marked PEP–TPP
and MitoTracker Green-labeled mitochondria was imaged by a confocal
microscope (Thermo Fisher Scientific). To observe the dynamic internalization
of transplanted mitochondria, PEP(Cy5)–TPP–mitochondria
was added into the culture medium. Then the fluorescence intensity
of MitoTracker Green and Cy5 was detected by the Lionheart FX living
cell imaging analysis system (BioTek, Winooski, VT, USA).
For
in vivo imaging, to detect the retention of transplanted PEP(Cy5)–TPP–mitochondria
(GFP), mouse hearts were imaged by a fluorescence detection system
(IVIS Lumina XRMS, USA) after reperfusion for 3, 6, and 24 h. Besides,
the Cox4i1-GFP marked mitochondria of formalin-fixed heart tissue
sections were imaged with a fluorescence microscope (Thermo Fisher
Scientific).
10
CM-H2DCFDA Fluorescence Imaging
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The cells were treated with 10 μM CM-H2DCFDA in the dark at 37°C for 30 min, and the excess probe was removed via washing. The samples were analyzed using a Fluorescence Microscope (Thermo Fisher Scientific, United States).
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