NADH autofluorescence was monitored using an epifluorescence inverted microscope equipped with a × 40 fluorite objective. Excitation light (350 nm) was provided by a Xenon arc lamp, the beam passing through a monochromator (Cairn Research, Kent, UK). Emitted fluorescence light was reflected through a 455-nm long-pass filter to a cooled CCD camera (Retiga, QImaging, Canada). Imaging data were collected and analyzed using software from Andor (Belfast, UK). FAD++ autofluorescence was monitored using a Zeiss 710 VIS CLSM and a × 40 objective. We used 454-nm laser for excitation with emission at 505–550 nm.
710 vis clsm
Manufactured by Zeiss
Sourced in Germany
The 710 VIS CLSM is a confocal laser scanning microscope designed for visible light microscopy applications. The core function of this equipment is to provide high-resolution, optical sectioning capabilities for imaging a variety of biological and material samples.
Automatically generated - may contain errors
Lab products found in correlation
Monochromator, by Cairn Research (4 mentions)
Xenon arc lamp, by Cairn Research (2 mentions)
Mitotracker red cm h2xros, by Thermo Fisher Scientific (1 mentions)
710 confocal microscope, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Volocity 3d image analysis software, by PerkinElmer (1 mentions)
13 protocols using 710 vis clsm
1
NADH and FAD++ Autofluorescence Imaging
2
Fluorescence Imaging of NADH and FAD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NADH autofluorescence was measured using an epifluorescence inverted microscope equipped with a 20X fluorite objective. Excitation light at a wavelength of 350 nm was provided by a Xenon arc lamp, the beam passing through a monochromator (Cairn Research, Kent, UK). Emitted fluorescence light was reflected through a 455 nm long-pass filter to a cooled CCD camera (Retiga, QImaging, Canada) and digitized to 12 bit resolution. Imaging data were collected and analysed using software from Andor (Belfast, UK). FAD autofluorescence was monitored using a Zeiss 710 VIS CLSM equipped with a META detection system and a 40x oil immersion objective. Excitation was using the 454 nm Argon laser line and fluorescence was measured from 505 to 550 nm. Illumination intensity was kept to a minimum (at 0.1–0.2% of laser output) to avoid phototoxicity and the pinhole set to give an optical slice of ~2 µm. FAD and NADH redox indexes and mitochondrial pools were estimated according the method described in Bartolome et al. [46] (link).
3
Mitochondrial Membrane Potential Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were loaded with 1 μM MitoTracker Red CM-H2Xros (Thermo Scientific) incubated for 20 min before the beginning of the experiments, and then imaged the increase of fluorescence over time using 561 nm excitation and long pass filter. MitoTracker Red CM-H2Xros is a reduced, non-fluorescent dye which fluoresces upon oxidation and enters the mitochondria according to the membrane potential. Confocal images were obtained using a Zeiss 710 VIS CLSM equipped with a META detection system and a 40× oil immersion objective.
4
Measuring Mitochondrial Membrane Potential
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tetramethyl rhodamine, methyl ester (TMRM) is a cationic, cell-permeant fluorescent dye readily sequestered by healthy mitochondria. Basal mitochondrial membrane potential was measured in the non-quench mode, loading the cells with 25 nM TMRM for 40 min, z-stacks were acquired with a 710 Zeiss Confocal microscope. To measure small differences in the maintenance of ΔΨm we used a de-quenching mode. Cells were incubated for 20 min with 1 μM TMRM. Depolarization, after oligomycin (2 μg/mL) administration, was detected as an increase of ΔΨm. At the end of each experiment, 1 μM Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) was applied. FCCP is protonophore that dissipates the potential completely. TMRM was visualized using an excitation wavelength of 561 nm with a long pass filter using a Zeiss 710 VIS CLSM equipped with a META detection system and a 40× oil immersion objective.
5
Confocal Imaging of Mitochondrial Membrane Potential
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confocal images were obtained using a Zeiss 710 VIS CLSM equipped with a META detection system and a ×40 oil immersion objective. Illumination intensity was kept to a minimum (at 0.1–0.2% of laser output) to avoid phototoxicity and the pinhole set to give an optical slice of ∼2 µm. TMRM was excited using the 560 nm laser line and fluorescence measured above 580 nm. All data presented were obtained from at least five coverslips and two to three different cell preparations for cell experiments, and six coverslips and at least six counts for fly brain experiments. The same anatomical area of the fly brain was used each time.
6
Measuring Mitochondrial Membrane Potential with TMRM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For measurements of ΔΨm, isolated mitochondria were plated on 22 mm glass coverslips and loaded for 30 min at room temperature with 50 nM tetramethylrhodamine methylester (TMRM; Invitrogen) in a medium containing: Tris HCl 10 mM; KCl 120 mM; EGТА 1 mM; glutamate 5 mM; malate 1 mM; KH2PO4 2.5 mM. Before imaging the media was replaced with 40 mM Ca(NO3)2 solution. The dye remained present in the media at the time of recording. Confocal images were obtained using a Zeiss 710 VIS CLSM equipped with a META detection system and a 40× oil immersion objective. TMRM was excited using the 560 nm laser line and fluorescence was measured above 580 nm. For analysis of response to mitochondrial toxins, images were recorded continuously from a single focal plane.
7
Quantifying Cellular Glutathione Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reduced Glutathione (GSH) was measured with monochlorobimane (MCB), a non-fluorescent compound that becomes fluorescent when binds GSH. Cells were incubated with 50 μM MCB for 30 min. Confocal images (z-stacks) were acquired for quantitation using a Zeiss 710 VIS CLSM equipped with a META detection system and a 40× oil immersion objective.
8
Measurement of NADH and FAD Autofluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NADH autofluorescence was measured using an epifluorescence inverted microscope equipped with a 20× fluorite objective. Excitation light at a wavelength of 350 nm was provided by a Xenon arc lamp, with the beam passing through a monochromator (Cairn Research, Faversham, Kent, UK). Emitted fluorescence light was reflected through a 455 nm long-pass filter to a cooled CCD camera (Retiga, QImaging, Surrey, BC, Canada) and digitised to 12 bit resolution. Imaging data were collected and analyzed using software from Andor (Belfast, UK). FAD autofluorescence was monitored using a Zeiss 710 VIS CLSM equipped with a META detection system and a × 40 oil immersion objective. Excitation was measured using the 454 nm Argon laser line and fluorescence was measured from 505 to 550 nm. Illumination intensity was kept to a minimum (at 0.1–0.2% of laser output) to avoid phototoxicity and the pinhole set to give an optical slice of ~2 μm.
9
Quantifying cellular redox state
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NADH autofluorescence was measured using an epifluorescence inverted microscope equipped with a ×20 fluorite objective. Excitation light at a wavelength of 350 nm was provided by a Xenon arc lamp, the beam passing through a monochromator (Cairn Research).
Emitted fluorescence light was reflected through a 455 nm long-pass filter to a cooled charge-coupled device (CCD) camera (Retiga; QImaging) and digitized to 12-bit resolution. Imaging data were collected and analyzed using software from Andor. Flavin adenine dinucleotide (FAD) autofluorescence was monitored using a Zeiss 710 VIS CLSM equipped with a META detection system and a ×40 oil immersion objective. Excitation was using the 454 nm Argon laser line and fluorescence was measured from 505 to 550 nm. Illumination intensity was kept to a minimum (at 0.1%-0.2% of laser output) to avoid phototoxicity and the pinhole set to give an optical slice of ∼2 µm. FAD and NADH redox indexes and mitochondrial pools were estimated according to previously described (Al-Menhali et al., 2020) (link).
Emitted fluorescence light was reflected through a 455 nm long-pass filter to a cooled charge-coupled device (CCD) camera (Retiga; QImaging) and digitized to 12-bit resolution. Imaging data were collected and analyzed using software from Andor. Flavin adenine dinucleotide (FAD) autofluorescence was monitored using a Zeiss 710 VIS CLSM equipped with a META detection system and a ×40 oil immersion objective. Excitation was using the 454 nm Argon laser line and fluorescence was measured from 505 to 550 nm. Illumination intensity was kept to a minimum (at 0.1%-0.2% of laser output) to avoid phototoxicity and the pinhole set to give an optical slice of ∼2 µm. FAD and NADH redox indexes and mitochondrial pools were estimated according to previously described (Al-Menhali et al., 2020) (link).
10
Measuring Mitochondrial Membrane Potential
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For measurements of ΔΨ m , cells plated on 25-mm glass coverslips were loaded for 30 min at room temperature (RT) with 25 nM tetramethylrhodamine methyl ester (TMRM; Invitrogen) in a HEPESbuffered saline solution (HBSS) composed of 156 mM NaCl, 3 mM KCl, 2 mM MgSO 4 , 1.25 mM KH 2 PO 4 , 2 mM CaCl 2 , 10 mM glucose, and 10 mM HEPES; pH adjusted to 7.35 with NaOH. The dye remained present in the media at the time of recording. Confocal images were obtained using a Zeiss 710 VIS CLSM equipped with a META detection system and a ×40 oil immersion objective. TMRM was excited using the 560 nm laser line and fluorescence was measured above 580 nm (M. H. R. Ludtmann et al., 2018) (link). For basal ΔΨ m measurements, Z-stack images were obtained by confocal microscopy and analyzed using Zeiss software. For analysis of response to mitochondrial toxins, images were recorded continuously from a single focal plane. TMRM is used in the redistribution mode to assess ΔΨ m , and, therefore, a reduction in TMRM fluorescence represents mitochondrial depolarization.
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!