Cellular Redox Profiling Using High-content Microscopy

Summary

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

Abstract

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 (ΔΨ_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 (ΔΨ_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.

Introduction

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^•-) 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 (Δψ_m), i.e., ....

Protocol

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

1. Prepare complete medium by supplementing Dulbecco'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 445 mL of DMEM.
2. Prepare HBSS-HEPES (HH) imaging buffer by supplementing Hank's Balanced Salt Solution (with magnesium and calcium, but wit....

Results

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 Δψ_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 concentrations of TBH.......

Discussion

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 experiments^19,20,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM)	ThermoFisher Scientific	T668
CM-H2DCFDA (General Oxidative Stress Indicator)	ThermoFisher Scientific	C6827
Dimethyl sulfoxide	Sigma)Aldrich	D8418
MatriPlate 96-Well Glass Bottom MicroWell Plate 630 µL-Black 0.17 mm Low Glass Lidded	Brooks life science systems	MGB096-1-2-LG-L
HBSS w/o Phenol Red 500 mL	Lonza	BE10-527F
DMEM high glucose with L-glutamine	Lonza	BE12-604F
Phosphate Bufered Saline (PBS) w/o Ca and Mg	Lonza	BE17-516F
HEPES 1 M 500 mL	Lonza	17-737F
Trypsin-Versene (EDTA) Solution	Lonza	BE17-161E
Cy3 AffiniPure F(ab')2 Fragment Donkey Anti-Rabbit IgG (H+L)	Jackson	711-166-152	Antibody used for acquiring flat-field image
Alexa Fluor 488 AffiniPure F(ab')2 Fragment Donkey Anti-Rabbit IgG (H+L)	Jackson	711-546-152	Antibody used for acquiring flat-field image
Name	Company	Catalog Number	Comments
Equipment
Nikon Ti eclipse widefield microscope	Nikon
Perfect Focus System (PFS)	Nikon		hardware-based autofocus system
CFI Plan Apo Lambda 20X objective	Nikon
Name	Company	Catalog Number	Comments
Software
NIS Elelements Advanced Research 4.5 with JOBS module	Nikon		This software is used to steer the microscope and program/perform the automatic image acquisition prototocol
ImageJ (FIJI) Version 2.0.0-rc-43/1.50g
RStudio Version 1.0.44	Rstudio
R version 3.3.2