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1. Rat hippocampal astrocyte primary culture
<ol>
	<li>Sacrifice five rat pups (Wistar IGS Rat) by decapitation at postnatal days 1-2.</li>
	<li>Remove the brain and keep it in a Petri dish containing 5 mL of fresh astrocyte medium (Dulbecco&#39;s Modified Eagle Medium or DMEM with GlutaMAX supplemented with 1% Penicillin/Streptomycin and 10% fresh Horse Serum).</li>
	<li>Isolate the hippocampus. Dissociate in 5 mL of the astrocytic medium by three passages through a 21 G needle and three passages through a 25 G needle.</li>
	<li>Transfer the dissociated cells to a 15 mL centrifuge tube and count in a hemocytometer.</li>
	<li>Plate 20,000-25,000 cells/cm2 in multiwell dishes (9.4 mm x 10.7 mm x 9.3 mm) and store them at 37 &#176;C under an atmosphere containing 5% CO2 for the rest of the experiment.</li>
	<li>Replace the medium entirely at 3 days in vitro (Division or DIV 3).</li>
	<li>At DIV 8, add 0.6 pg of p24 antigen per cell of lentiviral vector coding for mitochondrial biosensor MitoTimer (LV-G1-MitoTimer) diluted in phosphate-buffered saline (Phosphate-buffered saline or PBS + 0.01% Bovine serum albumin or BSA).</li>
	<li>At DIV 9, wash with pre-warmed 1x sterile PBS (37 &#176;C) and add fresh astrocyte medium.</li>
	<li>At DIV 11, 2 washes were performed with pre-warmed 1x sterile PBS (37 &#176;C), and fresh astrocyte medium was added without phenol red. 
	&#160;NOTE: The choice of the coating depends on the intended assay. As a standard coating for astrocyte primary culture, it is recommended to use 0.2 mg/mL poly-D-lysine or 8.7 &#181;g/cm2 of basement membrane matrix (e.g., Matrigel). To visualize the mitochondrial system of astrocytes, it is essential to work on flattened and stretched cells. Combining the basement membrane matrix on IBIDI u-slides is the most suitable in this context. It is also crucial not to work with phenol red, which is toxic for the cell under repeated exposure to light.</li>
</ol>
2. Long-term monitoring of the mitochondrial system.
<ol>
	<li>Assess the astrocytic mitochondrial system at least 3-5 days after the lentiviral infections with a lentiviral vector (LV-G1-MitoTimer) to allow a sufficient level of fluorescence in the mitochondria.</li>
	<li>Image the cells with an inverted microscope controlled by the acquisition and analysis software and a module for complete automation of acquisition. 
	NOTE: Refer to the Table of Materials for the details of the software and module used in this study.</li>
	<li>Ensure the microscope is equipped with a cage incubator (see Table of Materials) to maintain astrocyte culture at 5% CO2 and 37 &#176;C throughout the experiment.</li>
	<li>Capture fluorescence images using sequential excitation at 490 nm (for the green channel) and 550 nm (for the red channel), detecting green (500-540 nm for the green channel) and red (550-600 nm for the red channel) fluorescence signals.</li>
	<li>Select 5 astrocytes per well with a mitochondrial network expressing sufficient levels of LV-G1-MitoTimer using a 40x magnification. Take care to select astrocytes that are as flat and large as possible and not located in clusters of cells (to work on individual cells).</li>
	<li>Save the coordinates of the 5 selected cells in an xlm file (map.xlm). This map.xlm allows the user to return to the same cells over time.</li>
	<li>Acquire image sequences (1 image/s for 60 s) using a 150x magnification (100x oil immersion objective, 1.5x intermediate magnification) for each coordinate.</li>
	<li>Repeat image acquisition (1 image/s for 60 s) for the same astrocytes 6 h, 12 h, and 24 h after treatments. 
	NOTE: Use a microscope equipped with a fast and efficient autofocus system. In this context, the hardware solution Perfect Focus System (PFS) was used. PFS uses near-infrared 870-nm LED and CCD line sensors to combat axial focus fluctuations in real-time during long-term imaging investigations.</li>
</ol>
3. Analysis of individual mitochondrial morphology and LV-G1-MitoTimer ratio
NOTE: NIS General Analysis 3 (GA3) from Nikon was used to automatize morphometric analysis in this study.
<ol>
	<li>For each image sequence (baseline, 6 h, 12 h, 24 h), select the first frame for red and green channels by clicking on ND Processing &#62; Select Frame.</li>
	<li>Merge the red and the green channels by selecting Conversions &#62; Merge Channel.</li>
	<li>To correct image shading, select Preprocessing &#62; Auto Shading Correction.</li>
	<li>Apply the Rolling Ball algorithm by selecting Preprocessing &#62; Rolling Ball.</li>
	<li>To generate binary masks for each mitochondrion, select Segmentation &#62; Threshold.</li>
	<li>Remove any objects truncated by the border by selecting Binary processing &#62; Touching Border.</li>
	<li>To measure the surface area, select Measurement &#62; Object Area.</li>
	<li>To measure the diameter, select Measurement &#62; Eq Diameter.</li>
	<li>To measure the length, select Measurement &#62; Length.</li>
	<li>To measure the width, select Measurement &#62; Width.</li>
	<li>To measure roughness, select Measurement &#62; Roughness.</li>
	<li>To measure circularity, select Measurement &#62; Circularity.</li>
	<li>To measure elongation, select Measurement &#62; Elongation.</li>
	<li>Compose a group (right-click) with the above measurement and rename it MorphoData.</li>
	<li>Measure mean green intensity by selecting Measurement &#62; Mean Intensity.</li>
	<li>Measure mean red intensity by selecting Measurement &#62; Mean Intensity.</li>
	<li>To measure the Red/Green ratio, select Measurement &#62; Ratio.</li>
	<li>Compose a group (right-click) with the above measurement and rename it as RatioData.</li>
	<li>Export the table to a CSV file by selecting Reference &#62; Table to CSV.</li>
	<li>Save the GA3 script of analysis by selecting Save As 
	NOTE: A non-exhaustive list of the criteria used in this procedure is summarized in Figure 1, Table 1, and Table 2. The thresholds used were adapted to individualize as many mitochondria as possible (excluding large mitochondrial networks where possible). Wherever possible, mitochondrial networks are either individualized manually or excluded from the analyses. Due to intercellular variability, these analyses are performed on at least 20-25 cells per condition (with a minimum of 50 mitochondria by cell).</li>
</ol>
Table 1: Summary of selected parameters for mitochondrial morphology.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Morphology criteria</td>
			<td>Range</td>
			<td>Remarks</td>
		</tr>
		<tr>
			<td>Surface Area (SA)</td>
			<td>0.5&#8211;4 &#181;m2</td>
			<td rowspan="7">These criteria inform on the fragmented-elongated features of mitochondria. They generally evolve in the same direction. Fragmented mitochondria will have decreased surface area, diameter, length, and elongation factor, while roundness, sphericity, and width may be unchanged or increased.</td>
		</tr>
		<tr>
			<td>Diameter (D)</td>
			<td>0.5&#8211;1.5 &#181;m</td>
		</tr>
		<tr>
			<td>Length (L)</td>
			<td>0.5&#8211;5 &#181;m</td>
		</tr>
		<tr>
			<td>Width (W)</td>
			<td>0.5&#8211;2 &#181;m</td>
		</tr>
		<tr>
			<td>Roundness (R)</td>
			<td>0&#8211;1</td>
		</tr>
		<tr>
			<td>Sphericity (S)</td>
			<td>0&#8211;1</td>
		</tr>
		<tr>
			<td>Elongation factor (EF = L/W)</td>
			<td>1&#8211;10</td>
		</tr>
	</tbody>
</table>
Table 2: Summary of selected parameters for mitochondrial redox state.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Redox State criteria</td>
			<td>Range</td>
			<td>Remarks</td>
		</tr>
		<tr>
			<td>Individual ratio (G/R)</td>
			<td>0&#8211;10</td>
			<td rowspan="3">The ratio indicates the result of the redox state. It informs about the general state and age of the mitochondria in the cell. It is essential to consider that this ratio is the balance of biogenesis and degradation of mitochondria and the fission/fusion of oxidized mitochondria with reduced mitochondria. Therefore, evaluating the number of green and red puncta can powerfully help interpret the results.&#160; Green puncta mitochondria are determined when the intensity of green is 10 times that of red. Red puncta mitochondria are determined when the intensity of red is 10 times greater than green&#39;s. The redox state of an astrocyte is the average of all the mitochondria ratios of that cell.</td>
		</tr>
		<tr>
			<td>Percentage of green puncta mitochondria (Gpuncta)</td>
			<td>0%&#8211;100%</td>
		</tr>
		<tr>
			<td>Percentage of red puncta mitochondria (Rpuncta)</td>
			<td>0%&#8211;100%</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#181;-Slide 8 Well</td>
			<td>IBIDI</td>
			<td>80807</td>
			<td></td>
		</tr>
		<tr>
			<td>19 G needle</td>
			<td>Plexus SANTE</td>
			<td>PL001213</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needle</td>
			<td>Plexus SANTE</td>
			<td>PL000142</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G needle</td>
			<td>Plexus SANTE</td>
			<td>PL000133</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovin Serum Albumin</td>
			<td>LIFE TECH</td>
			<td>15260037</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>HAMAMATSU</td>
			<td>ORCA-flash4.0 V3 - C13440-20CU</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX(TM)</td>
			<td>THERMOFISHER</td>
			<td>61965059</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax Supplement</td>
			<td>THERMOFISHER</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>SIGMA</td>
			<td>16050122</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens</td>
			<td>Nikon Instruments</td>
			<td>CFI Plan Fluor 100x Oil</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Engines</td>
			<td>LUMENCOR</td>
			<td>SPECTRA X</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear-encoded motorized platine</td>
			<td>Nikon Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Instruments</td>
			<td>ECLIPSE Ti2-E MICROSCOPE INVERSE</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Stage Incubator with 3-channel manual gas mixer and gas bubbler/ humidity module</td>
			<td>OKOLAB</td>
			<td>H201-NIKON-TI-S-ER</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x liquid</td>
			<td>THERMOFISHER</td>
			<td>20012068</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>SIGMA</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 100 mm</td>
			<td>SIGMA</td>
			<td>P5731</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 35 mm</td>
			<td>SIGMA</td>
			<td>CLS430165</td>
			<td></td>
		</tr>
		<tr>
			<td>Pregnant Rats</td>
			<td>CHARLES RIVERS</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>Software Nikon NIS-HC</td>
			<td>Nikon Instruments</td>
			<td>NIS-Elements HC</td>
			<td></td>
		</tr>
		<tr>
			<td>Sofware Prism</td>
			<td>GraphPad</td>
			<td>V8.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup 500 mL</td>
			<td>MERCK MILLIPORE</td>
			<td>10412701</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence-based monitoring of mitochondrial stress in astrocytes

Source: Espourteille, J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62957">Live-imaging of Mitochondrial System in Cultured Astrocytes.</a> J. Vis. Exp. (2021)
This video demonstrates the evaluation of mitochondrial health in genetically modified astrocytes, which express a fluorescent protein within their mitochondria. The protein helps to track changes in mitochondrial health as its fluorescence changes from green to red, indicating stress or damage. Images are captured and analyzed using various parameters.

<img alt="Figure 1" src="/files/ftp_upload/22981/22981_Figure1.jpg" /> 
Figure 1: Astrocyte culture expressing LV-G1-MitoTimer biosensor. (A) Confocal photographs of astrocytes expressing LV-G1-MitoTimer. (B) Selection of confocal photographs of reduced (green), balanced (orange), and oxidized (red) mitochondria with different levels of fragmentation. (C) Summary diagram of the different criteria available for analysis in an astrocyte expressing LV-G1-MitoTimer. Scale bar: (A) Upper panel: 50 &#181;m, lower panel: 10 &#181;m, (B) 1 &#181;m.

Fluorescence-Based Monitoring of Mitochondrial Stress in Astrocytes

1. Rat hippocampal astrocyte primary culture

	Sacrifice five rat pups (Wistar IGS Rat) by decapitation at postnatal days 1-2.
	Remove the brain and keep ...

Begin with an astrocyte culture transduced to express a Mito-timer protein localized to their mitochondria.
Mito-timer acts as a mitochondrial biosensor, emitting green fluorescence in healthy mitochondria and transitioning to red fluorescence in stressed or damaged ones.
Microscopically select large and isolated astrocytes for imaging.
Using different excitation wavelengths, capture images for green and red fluorescence.
To analyze the images, select frames for both red and green channels, then choose merge channels.
Apply auto shading correction and run the image enhancement algorithm to enhance image clarity.
Select the desired morphological parameters of mitochondria for analysis.
Using the mean intensity tab, measure the mean intensities of red and green fluorescence.
Calculate the ratio between red and green intensities, which indicates the overall mitochondrial health of the astrocyte.

fluorescence-based-monitoring-of-mitochondrial-stress-in-astrocytes

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Infectious Diseases

31 Views. Source: Espourteille, J., et al. Live-imaging of Mitochondrial System in Cultured Astrocytes. J. Vis. Exp. (2021) This video demonstrates the evaluation of mitochondrial health in genetically modified astrocytes, which express a fluorescent protein within their mitochondria. The protein helps to track changes in mitochondrial health as its fluorescence changes from green to red, indicating stress or damage. Images are captured and analyzed using various parameters. 

Video: Fluorescence-Based Monitoring of Mitochondrial Stress in Astrocytes - Concept

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...

JoVE Journal

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Biology

Imaging of Mitochondrial Peroxide in Living Yeast Cells Using mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

Author Spotlight: Advancing Mitochondrial Research - mtHyper7 Biosensor for Subcellular Analysis 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...

Fluorescence-Based Monitoring of Mitochondrial Stress in Astrocytes

Transcript

Fluorescence-Based Monitoring of Mitochondrial Stress in Astrocytes

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells