Name: cellular redox profiling using high-content microscopy
Uploaded: 2017-05-14T13:00:00
Description: Watch this Scientific Journal Video about Cellular Redox Profiling Using High-content Microscopy at JoVE.com

This research was supported by the University of Antwerp (TTBOF/29267, TTBOF/30112), the Special Research Fund of Ghent University (project BOF/11267/09), NB-Photonics (Project code 01-MR0110) and the CSBR (Centers for Systems Biology Research) initiative from the Netherlands Organization for Scientific Research (NWO; No: CSBR09/013V). Parts of this manuscript have been adapted from another publication1, with permission of Springer. The authors thank Geert Meesen for his help with the widefield microscope.

The protocol below is described as performed for NHDF cells and with use of the multiwell plates specified in the materials file. See Figure 1 for a general overview of the workflow.
1. Preparation of Reagents
<ol>
	<li>Prepare complete medium by supplementing Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% v/v Fetal Bovine Serum (FBS) and 100 IU/mL penicillin and 100 IU/mL streptomycin (PS). For 500 mL complete medium, add 50 mL of FBS and 5 mL of PS to 44...

<ol>
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This paper describes a high-content microscopy method for the simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction in NHDF. Its performance was demonstrated with a case study on SQV-treated NHDF. The results support earlier evidence from literature in which increased ROS levels or mitochondrial dysfunction have been observed after treatment with type 1 HIV protease inhibitors, albeit in separate experiments19,20,<sup...

The concentration of intracellular ROS is meticulously regulated through a dynamic interplay between ROS producing and ROS defusing systems. Imbalance between the two provokes a state of oxidative stress. Among the major sources of ROS are mitochondria1. Given their role in cellular respiration, they are responsible for the bulk of intracellular superoxide (O2&#8226;-) molecules2. This mostly results from electron leakage to O2 at complex 1 of the electron transport chain under conditions of strong negative inner mitochondrial membrane potential (&#916;&#968;m), i.e., mitochondrial hyperpolarization. On the other hand, mitochondrial depolarization has also been correlated with increased ROS production pointing to multiple modes of action3,4,5,6,7,8. Furthermore, through redox modifications in proteins of the fission-fusion machinery, ROS co-regulate mitochondrial morphology9.For example, fragmentation is correlated with increased ROS production and apoptosis10,11, while filamentous mitochondria have been linked to nutrient starvation and protection against mitophagy12. Given the intricate relationship between cellular ROS and mitochondrial morphofunction, both should ideally be quantified simultaneously in living cells. To do exactly this, a high-content imaging assay was developed based on automated widefield microscopy and image analysis of adherent cell cultures stained with the fluorescent probes CM-H2DCFDA (ROS) and TMRM (mitochondrial &#916;&#968;m and morphology). High-content imaging refers to the extraction of spatiotemporally rich (i.e., large number of descriptive features) information about cellular phenotypes using multiple complementary markers and automated image analyses. When combined with automated microscopy many samples can be screened in parallel (i.e. high-throughput), thereby increasing the statistical power of the assay. Indeed, a main asset of the protocol is that it allows for simultaneous quantification of multiple parameters in the same cell, and this for a large number of cells and conditions.
The protocol is divided into 8 parts (described in detail in the protocol below): 1) Seeding cells in a 96-well plate; 2) Preparation of stock solutions, working solutions and imaging buffer; 3) Setting up of the microscope; 4) Loading of the cells with CM-H2DCFDA and TMRM; 5) First live imaging round to measure basal ROS levels and mitochondrial morphofunction; 6) Second live imaging round after addition of tert-butyl peroxide (TBHP) to measure induced ROS levels; 7) Automated image analysis; 8) Data Analysis, Quality Control and Visualization.
The assay was originally developed for normal human dermal fibroblasts (NHDF). Since these cells are large and flat, they are well suited for assessing mitochondrial morphology in 2D widefield images13,14. However, with minor modifications, this method is applicable to other adherent cell types. Moreover, next to the combination of CM-H2DCFDA and TMRM, the workflow complies with a variety of fluorescent dye pairs with different molecular specificities1,15.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM)</td>
			<td>ThermoFisher Scientific</td>
			<td>T668</td>
			<td></td>
		</tr>
		<tr>
			<td>CM-H2DCFDA (General Oxidative Stress Indicator)</td>
			<td>ThermoFisher Scientific</td>
			<td>C6827</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma)Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>MatriPlate 96-Well Glass Bottom MicroWell Plate 630 &#181;L-Black 0.17 mm Low Glass Lidded</td>
			<td>Brooks life science systems</td>
			<td>MGB096-1-2-LG-L</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS w/o Phenol Red 500 ml</td>
			<td>Lonza</td>
			<td>BE10-527F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose with L-glutamine</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Bufered Saline (PBS) w/o Ca and Mg</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1M 500mL</td>
			<td>Lonza</td>
			<td>17-737F</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-Versene (EDTA) Solution</td>
			<td>Lonza</td>
			<td>BE17-161E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure F(ab&#39;)&#8322; Fragment Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson</td>
			<td>711-166-152</td>
			<td>Antibody used for acquiring flat-field image</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure F(ab&#39;)&#8322; Fragment Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson</td>
			<td>711-546-152</td>
			<td>Antibody used for acquiring flat-field image</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Ti eclipse widefield microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perfect Focus System (PFS)</td>
			<td>Nikon</td>
			<td></td>
			<td>hardware-based autofocus system</td>
		</tr>
		<tr>
			<td>CFI Plan Apo Lambda 20x objective</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elelements Advanced Research 4.5 with JOBS module</td>
			<td>Nikon</td>
			<td></td>
			<td>This software is used to steer the microscope and program/perform the automatic image acquisition prototocol</td>
		</tr>
		<tr>
			<td>ImageJ (FIJI) Version 2.0.0-rc-43/1.50g</td>
			<td></td>
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cellular redox profiling using high-content microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

The assay has been benchmarked using several control experiments, the results of which are described in Sieprath et al.1. In brief, the fluorescence response of CM-H2DCFDA and TMRM to extraneously induced changes in intracellular ROS and &#916;&#968;m, respectively has been quantified to determine the dynamic range. For CM-H2DCFDA, NHDF showed a linear increase in fluorescence signal when treated with increasing concentrati...

Watch this Scientific Journal Video about Cellular Redox Profiling Using High-content Microscopy at JoVE.com

Cellular Redox Profiling Using High-content Microscopy

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

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