Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Summary

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

Abstract

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H₂O₂ production, respectively.

Introduction

Reactive oxygen species (ROS) are involved in a wide variety of biological processes such as host defense, signaling, tissue development and response to injury as well as hypertension and cancer. The well-studied phagosomal NADPH oxidase machinery is dedicated to rapid ROS generation, known as the oxidative burst, to kill bacteria ingested in neutrophils' phagosomes¹. In addition, leakage of electrons as a byproduct of the mitochondrial respiratory chain was previously thought to be responsible only for an unregulated source of ROS. But recently, it was identified as an important mechanism to contribute to intraphagosomal killing of bacteria in mouse macrophages². In recent years, the social amoeba, Dictyostelium, has become a powerful and popular model to study cell intrinsic mechanisms of the innate immune response. Indeed, Dictyostelium and human phagocytes share a surprisingly high level of conservation in molecular machineries responsible for bacteria sensing, engulfment and killing^3,4. The homologs of proteins and enzymes related to ROS production or detoxification, such as NADPH oxidases, catalases, superoxide dismutases, can be found in both human and Dictyostelium. As the best studied amoeba, Dictyostelium presents several unique advantages over mammalian model system. They grow at room temperature without the need for CO₂, with a doubling time of 8-10 hr. They can be easily kept as adherent or suspension cultures. In addition, thanks to their fully sequenced and annotated haploid genome, and to easy genetic manipulation, Dictyostelium has become a very attractive experimental model organism.

In previous studies, various chlorinated and fluorinated derivatives of fluorescein (collectively known as OxyBurst Green, OBG) that emit fluorescence after oxidation by ROS, have been used as a ROS reporter. Cell-permeant esterified derivatives are used to measure cytoplasmic ROS, whereas cell-impermeant dextran- or protein-coupled derivatives are used to measure extracellular ROS. In particular, OBG BSA-coated beads have already been used to detect phagosomal ROS production in mammalian cells⁵. However, the microplate reader-based approach can only give an averaged ROS generation curve from a population of cells. With the present protocol, by using Dictyostelium , a professional phagocyte, and optimized experimental conditions, we obtain stable and efficient phagocytosis without conjugating any opsonin onto the beads. DHE has been used in various model systems, such as mammalian neutrophils and macrophages, to detect ROS production^6-9. Meanwhile, there are some controversies over the specificity and sensitivity of the method^8,10. As an improved version of Amplex Red, the fluorescence intensity of AUR is less sensitive to pH, which makes it more suitable to measure ROS in nonneutral or weakly acidic milieus. AUR has been recently applied in several mammalian systems^11,12, but its use in nonmammalian models, which quite often require weakly acidic growth media, has not been reported yet. In addition, there is no published protocol to quantitatively and dynamically measure ROS production and localization in the social amoeba Dictyostelium.

The goal of the presented set of protocols is to provide an easy and versatile solution to monitor various ROS and their localization, and further give insights into ROS-related cellular mechanisms. For the OBG assay, we used live microscopy to monitor the whole process of phagosomal ROS generation after uptake of OBG-coated beads by Dictyostelium cells, and provided a new approach to study the mechanism of intraphagosomal killing of bacteria. We have optimized medium-throughput DHE and AUR assays in Dictyostelium, to measure intracellular superoxide and extracellular H₂O₂ production, respectively, by using LPS as a potent ROS stimulator. Note that LPS was recently shown to increase the bactericidal activity of Dictyostelium towards phagocytosed bacteria¹³. In addition, treatment with DEDTC and catalase explicitly confirmed that these two methods specifically measure different types and subcellular localizations of ROS in Dictyostelium. Finally, the OBG assay can be adapted to any adherent phagocytic cell, by further opsonizing the beads with ligands for phagocytic receptors of animal cells. In principle, the DHE and AUR assays can also be fine-tuned for measurements in any adherent or nonadherent cell under appropriate experimental conditions.

Protocol

1. Visualization and Qualitative Measurement of ROS Production in Phagosomes

The OBG-coated beads should be prepared in advance. The beads coating procedures and agar overlay technique are adapted from published references^5,14.

1. Add 1 ml suspension of 3.0 μm carboxylated silica beads (about 1.8 x 10⁹ beads) into an 1.5 ml tube, wash the beads 3x with 1 ml of PBS by quick spin and vortexing.
2. Resuspend the beads in 1 ml of PBS containing 25 mg/ml cyanamide (to activate the carboxylated silica beads to covalently bind prelabeled BSA), incubate on a wheel for 15 min.
3. Wash 3x with 1 ml of coupling buffer (0.1 M sodium borate, pH 8.0) by quick spin (centrifuge at full speed in a table top mini centrifuge for 5 sec or alternatively centrifuge at 2,000 x g for 1 min in a table top centrifuge) and vortexing to remove excess cyanamide.
4. Mix the washed beads with 500 μl of coupling buffer containing 1 mg of OxyBurst Green (OBG) H2HFF (dihydro-2',4,5,6,7,7'-hexafluorofluorescein)-BSA, fill the tube with nitrogen gas from a standard gas-can before capping, and incubate on a wheel for 14 hr (overnight) at RT in the dark.
5. Wash the beads 2x with 1 ml of quenching buffer (250 mM glycin in PBS) to remove unreacted OBG, and twice with coupling buffer to remove the quenching buffer by quick spin and vortexing.
6. Add 1 ml of coupling buffer containing 50 μg of Alexa fluor 594 succinimidyl ester to conjugate to BSA. Fill the tube with nitrogen before capping, and incubate on a wheel for 1.5 hr at room temperature in the dark.
7. Wash the beads 3x with 1 ml of quenching buffer by quick spin and vortexing to stop the reaction, then wash the beads 3x with 1 ml of PBS by quick spin and vortexing.
8. Finally, resuspend the beads in 1 ml of PBS with 2 μl of 10% w/v azide for long-term storage. Measure the concentration of beads in the suspension with a hemocytometer (usually around 1-2 x 10⁹ beads/ml), fill the tube with nitrogen before capping, and store at 4 °C in the dark.
9. The coating efficiency of the OBG fluorescein can be tested by checking the emission spectrum using excitation at 500 nm. When oxidized by H₂O₂ in the presence of horseradish peroxidase (HRP), the fluorescence intensity of oxidized beads, at emission peak (538 nm), is 11-12x higher than that of nonoxidized coated beads.
10. Harvest exponentially growing Dictyostelium cells from a 10 cm Petri dish, plate different densities of cells on 3 cm dishes with an optically clear plastic or glass bottom, and grow them overnight. Choose the dishes that are about 80% confluent for the experiment. A detailed protocol for cultivating Dictyostelium cells has been published¹⁵.
11. Replace the culture medium with LoFlo medium (LF medium), incubate for 2 hr before the experiment in order to decrease the extracellular and intraendosomal autofluorescence of the HL5C culture medium.
12. In parallel, melt 10 ml 1.5% bacto agar in LF medium, pour the agar onto a flat surface (i.e. a glass plate of 10 cm x 10 cm) in order to form an agar layer about 1 mm thick, wait for 10-15 min to solidify. Cut the agar layer into 2 cm x 2 cm squares and place in LF medium for later use.
13. Meanwhile, prepare the spinning-disk or wide field microscope, set the temperature of the environmental chamber at 22 °C and adjust the settings for this experiment. (See additional comments in the discussion).
14. After 2 hr of incubation, aspirate the LF medium from the 3 cm dish, but the cell monolayer should still be covered by a thin film of medium. Dilute the coated beads to 1.5 x 10⁷ beads/ml, and add 10 μl onto the cell layer.
15. Take one square agar sheet, drain excess liquid but keep wet. Gently put the agar square on top of the cell layer. Do not move the agar square when it is lying on the cells. The agar overlay is used to increase contact between beads and cells, thereby improving uptake, and it also slightly compresses the cells, keeping them better in the focal plane of the objective.
16. Place the lid onto the dish, place it on the microscope stage, and automatically take pictures in the red, green and phase channels every 1 min for 2 hr or longer.
17. Select and focus on cellular events that contain the whole process of phagocytosis. Merge the optimized 3 channels and assemble the pictures into a movie using professional image processing software. Quantify fluorescence intensities of each selected beads in red and green channels, and the ratio of green/red will reflect the dynamic phagosomal ROS production of the cells.

2. Quantitative and Medium-throughput Measurement of Intracellular Superoxide Production

1. Collect one 80% confluent dish of Dictyostelium in 10 ml of HL5C medium. Centrifuge Dictyostelium cells at 850 x g for 5 min, carefully and completely aspirate excess medium. Resuspend cells in SS6.4 buffer [0.12 M sorbitol in Sorensen buffer (14.69 mM KH₂PO₄ and 6.27 mM Na₂HPO₄), pH 6.4], count cells and dilute to a final density of 6 x 10⁶ cells/ml (see additional comments in the discussion).
2. Add 50 μl of cell suspension into each well of a white nontransparent 96 well plate (see additional comments in the discussion).
3. Dilute DHE stock (30 mM in DMSO) 500 fold with SS6.4, pipette 50 μl of diluted DHE into each well using a multichannel pipette if necessary. The final concentration of DHE is 30 μM. The reaction starts immediately.
4. Stimuli or inhibitors can be added at this point, or at other time points according to specific needs.
5. Use the end point "top reading" mode of a fluorescence microplate reader, with fluorescence excitation/emission at 522 nm/605 nm, read every 2 min for 1 hr, medium shake 5 sec/min, at 22 °C.

3. Quantitative and High Throughput Measurement of Extracellular H₂O₂ Production

1. Follow the same steps as described in 2.1 and 2.2.
2. Prepare diluted HRP by adding 5 μl of HRP stock (100 U/ml in distilled water) into 10 ml of SS6.4, vortex or invert the tube to mix well and keep on ice for later use. The diluted HRP solution is at 0.05 U/ml.
3. Prepare the AUR reaction mixture by mixing the AUR stock (10 mM AUR in DMSO) and the diluted HRP solution into SS6.4 buffer to the final concentrations of 6.25 μM, and 0.005 U/ml, respectively. As an example, to prepare the reaction mixture for 40 reactions, mix 1600 μl of SS6.4 with 400 μl of diluted HRP and 2.5 μl of AUR stock solution.
4. Add 50 μl of AUR mixture into each well using a multichannel pipette if necessary. Now the reaction starts.
5. Stimuli or inhibitors can be added at this point, or at other time points according to specific needs.
6. Use the end point "top reading" mode of a fluorescence micro plate reader, with fluorescence excitation/emission at 530 nm/590 nm, read every 2 min for 1 hr, medium shake 5 sec/min, at 22 °C.

Results

The generation of ROS in phagosomes can be visualized qualitatively and dynamically by microscopy (File S1). The red fluorescence emitted by Alexa fluor 594 is pH-insensitive and remains constant in the phagosomal environment, while oxidation of OBG increases its fluorescence in the green channel. The emission spectra of in vitro oxidized and nonoxidized OBG-coated beads are compared in Figure 1B, showing a significant increase in intensity after oxidation. To facilitate visuali...

Discussion

Compared to the previously described methods, the OBG assay dynamically visualizes the process of phagosomal ROS generation at the single cell level, instead of measuring an average ROS signal from a population of cells. Such population-averaging methods tend to obscure critical information caused by nonsynchronous phagocytosis. We successfully adapted DHE and AUR assays to Dictyostelium and optimized the protocols. Most importantly, we specified the types and subcellular localizations of ROS measured by these t...

Materials

Name	Company	Catalog Number	Comments
REAGENTS
OxyBURST Green H2HFF BSA	Invitrogen	O-13291
Alexa Fluor 594	Invitrogen	A-20004
Carboxylated silica beads	Kisker Biotech	PSi-3.0COOH
HL5C medium	ForMedium	HLC0102
LoFlo medium	ForMedium	LF1001
Bacto agar	BD	204010
Dihydroethidium	Sigma	37291-25MG
Amplex UltraRed	Invitrogen	A36006
Horseradish peroxidase	Roche	10108090001
Diethyldithiocarbamate (DEDTC)	Sigma	D3506-100G
Catalase	Sigma	C9322-1G
Cyanamide	Sigma	187364-25G
Lipopolysaccharides	Sigma	L2630-25G
EQUIPMENT
ibidi μ-Dish 35 mm, high	ibidi	81156	Bottom made of optically clear plastic
MatTek dish 35 mm	MatTek corporation	P35G-1.5-14-C	Bottom made of a glass coverslip
Scepter Cell Counter	Merck Millipore	PHCC00000
White 96 well plate	Nunc	236108
Centrifuge	SORVALL	Legend RT
Microplate reader	BioTek	Synergy Mx