Using redox-sensitive fluorescent probes to record real-time reactive oxygen species production in cells from mouse carotid body slices

Protocol Using redox-sensitive fluorescent probes to record real-time reactive oxygen species production in cells from mouse carotid body slices Reactive oxygen species (ROS) are important signaling molecules for physiologic processes such as acute response to hypoxia. However, reliable real-time ROS measurement in cells has been a long-standing methodological challenge. Here, we present a protocol to record acute changes in ROS production in sensory cells from mouse carotid body (CB) slices using redox-sensitive green fluorescent protein probes and microfluorimetry. This protocol provides sensitive and reproducible quantification of ROS during acute hypoxia in different subcellular compartments of CB glomus cells. Lin Gao, Ignacio Arias-Mayenco, Patricia Ortega-Sa´enz,Jose´ Lo´ pez-Barneo [email protected] Highlights ROS are signaling molecules in the carotid body (CB) during acute hypoxia Fluorescent roGFP probes are used to monitor acute changes of ROS in mouse CB slices Genetically encoded roGFP probes target different mitochondrial compartments Cellular basal redox status can be determined using roGFP targeted to cytosol Gao et al., STAR Protocols 2, 100535 June 18, 2021 ª2021 The Author(s). https://doi.org/10.1016/ j.xpro.2021.100535 ll OPEN ACCESS Protocol Using redox-sensitive fluorescent probes to record realtime reactive oxygen species production in cells from mouse carotid body slices Lin Gao, 1,2,3 Ignacio Arias-Mayenco, 1 Patricia Ortega-Sa ´enz, 1,2,3 and Jose ´Lo ´pez-Barneo 1,2,3,4,5, * 1 Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocı ´o/CSIC/Universidad de Sevilla, 41013 Seville, Spain 2 Departamento de Fisiologı ´aMe ´dica y Biofı ´sica, Facultad de Medicina, Universidad de Sevilla, 41013 Seville, Spain 3 Centro de Investigacio ´n Biome ´dica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031 Madrid, Spain 4 Technical contact 5 Lead contact *Correspondence: [email protected] https://doi.org/10.1016/j.xpro.2021.100535 SUMMARY Reactive oxygen species (ROS) are important signaling molecules for physiologic processes such as acute response to hypoxia. However, reliable real-time ROS measurement in cells has been a long-standing methodological challenge. Here, we present a protocol to record acute changes in ROS production in sensory cells from mouse carotid body (CB) slices using redox-sensitive green fluorescent protein probes and microfluorimetry. This protocol provides sensitive and reproducible quantification of ROS during acute hypoxia in different subcellular compartments of CB glomus cells. For complete details on the use and execution of this protocol, please refer to Ferna ´ndez-Agu¨era et al. (2015) and Arias-Mayenco et al. (2018). BEFORE YOU BEGIN Solution preparation Timing: 30 min See ‘‘materials and equipment’’ for a complete list of recipes for each solution. 1. Modified 0 Ca 2+ Tyrode’s solution, which should be kept at 4C up to 1 week until use. 2. Low-melting point agarose solution, which needs to be prepared the same day of CB dissection and sectioning. 3. Enzymatic digestion solution, which needs to be prepared fresh. 4. Culture medium, which needs to be prepared the same day of experiment, equilibrated in a cell culture incubator with 5% CO 2 at 37C for at least 1 h before use, and maintained at the cell culture incubator until use. 5. Recording solution, which is kept at 4Cupto1weekandpre-warmedto37 C before recording. Vibratome calibration Timing: 2 min STAR Protocols 2, 100535, June 18, 2021 ª2021 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1 ll OPEN ACCESS 6. Calibrate the vertical deflection of the blade (Z axis of the vibratome) using the Vibrocheck measurement device (Leica), following the manufacturer’s instruction. This can minimize vibration at this direction during sectioning, retaining viable cells on the section surface, where ROS production will be recorded. Note: Researchers using vibratome from other manufacturer should consult with the company for the availability of a calibration tool similar to Vibrocheck measurement device (Leica). Preparation of roGFP probes in aliquots Timing: 5 min 7. Aliquot roGFP probes, which have been incorporated into adenovirus vector, and store the aliquots at 80Cupto1year. CRITICAL: To obtain optimal transduction efficiency, aliquots of roGFP-containing adenovirus should be prepared on ice and dark, and then kept at 80C. Repeated freezingthawing should be avoided. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and virus strains VQAd ro-GFP ViraQuest Cat#111414 VQAd CMV GDP-roGFP ViraQuest Cat#022411 VQAd CMV mito-roGFP ViraQuest Cat#122909 Chemicals, peptides, and recombinant proteins Sodium thiopental Braun 8470006355732 Low melting-point agarose FMC BioProducts 50072 Phosphate-buffered saline Gibco 10010-015 Cyanoacrylate Loctite 8410436130431 Dithiothreitol Sigma D0632 Hydrogen peroxide Sigma H-1009 MitoTracker (Deep Red FM) Invitrogen 12010156 HEPES Sigma H-4034 Collagenase II Sigma C6885 Trypsin Sigma T8003 Porcine elastase Millipore 324682 DMEM (0 glucose) Gibco 11966-025 DMEM/F-12 Gibco 21331-020 Penicillin/Streptomycin Gibco 15140-122 L-glutamine Gibco 25030-024 Fetal bovine serum Gibco 10270-106 Insulin Novo Nordisk Atrapid, EU/1/02/230/011 Erythropoietin Sandoz Binocrit, EU1/07/410/028 Experimental models: Organisms/strains Wild-type C57BL/6 mouse, 8-week-old, male and female Charles River n/a Other Vibratome Leica VT1000S Direct microscope Olympus BX51 603/0.90w objective Olympus LUMPlanFI/IR (Continued on next page) ll OPEN ACCESS 2STAR Protocols 2, 100535, June 18, 2021 Protocol MATERIALS AND EQUIPMENT Modified 0 Ca 2+ Tyrode’s solution: Low-melting point agarose solution: Enzymatic digestion solution: Culture medium: Reagent Final concentration Amount NaCl (MW 58.44) 148 mM 1.73 g KCl (MW 74.55) 2 mM 0.03 g MgCl 2 (1M) 3 mM 0.6 mL HEPES (MW 238.3) 10 mM 0.477 g Glucose (MW 180.16) 10 mM 0.36 g ddH 2 O - up to 200 mL pH 7.4 Continued REAGENT or RESOURCE SOURCE IDENTIFIER 150 W Xenon lamp Ushio Inc. (Japan) PX45 Monochromator T.I.L.L. Photonics Polychrome V CCD camera Andor Technology iXon3 Live Acquisition software T.I.L.L. Photonics n/a Dichroic Semrock Di02-R488 Band-pass filter Semrock FF01-520/35 Gas mixture (5% CO 2 and 95% N 2 ) Air Liquide Espan ˜a, Spain Mixture DOC45 Gas mixture (20% O 2 ,5%CO 2 , and 75% N 2 ) Air Liquide Espan ˜a, Spain Mixture DOC45R Reagent Final concentration Amount Low-melting point agarose 1% (w/v) 30 mg Phosphate-buffered saline 13up to 3 mL/animal pH 7.4 Reagent Final concentration Amount CaCl 2 (5 mM) 50 mM30mL Collagenase II (60.24 mg/mL) 0.6 mg/mL 30 mL Trypsin (26.7 mg/mL) 0.27 mg/mL 30 mL Porcine elastase (125 U/mL) 1.25 U/mL 30 mL Phosphate-buffered saline 13up to 3 mL pH 7.4 Reagent Final concentration Amount DMEM (0 glucose)/DMEM-F12 (3:1) - up to 10 mL Penicillin (10000 U/mL/ Streptomycin (1 mg/mL) 100 U/mL / 10 mg/mL 100 mL L-glutamine (200 mM) 2 mM 100 mL (Continued on next page) ll OPEN ACCESS STAR Protocols 2, 100535, June 18, 2021 3 Protocol Note: Insulin and erythropoietin are added to culture medium, in addition to other supplements. In our experience, CB slices are healthier when cultured with medium supplemented with insulin and erythropoietin. In general, the appearance of glomeruli in the surface of the slice and secretory responses to stimuli are favored by this treatment. Recording solution: STEP-BY-STEP METHOD DETAILS Acute cellular and systemic responses are essential for mammals to adapt to environmental, physiological, and pathophysiological conditions causing oxygen (O 2 ) deficiency (hypoxia). The carotid body (CB), located in the carotid bifurcation, is the main arterial chemoreceptor responsible for detecting decreases in blood O 2 tension (hypoxemia) in order to trigger hyperventilation and sympathetic activation. The CB is organized in clusters of cells (glomeruli) and each glomerulus is composed of O 2 -sensitive glomus cells and other cell types. Recently, a signaling role of reactive oxygen species (ROS) has been reported underlying molecular mechanisms of acute O 2 sensing (Ortega-Sa´enz and Lo´ pez-Barneo, 2020). These findings would not have been possible without the development of redox-sensitive green fluorescent protein (roGFP) probes (Dooley et al., 2004;Cannon and Remington, 2008;Waypa et al., 2010). roGFP probes contain engineered cysteine residues, which form disulfide bonds in the presence of oxidants. This causes reciprocal changes in emission intensity when excited at two different wavelengths (see review by Cannon and Remington, 2008 for detailed biophysical properties of roGFPs). roGFP probes can react with several natural and synthetic redox reagents (Dooley et al., 2004), but are highly sensitive to H 2 O 2 , which is produced in mitochondria in the presence of superoxide dismutase. Using genetic targeting sequences, roGFP probes can be expressed in different intracellular compartments. These ratiometric redox probes allow us to detect rapid changes in intracellular redox status, with a time course of s/min within the dynamic range of the cellular (transmitter release) and systemic (increase in ventilation) physiological responses to hypoxia. In the current protocol, step-by-step method details are presented from CB sample preparation, roGFP probe transduction, to real-time measurement of ROS production by microfluorimetry. This method helps understand molecular mechanisms of acute O 2 sensing by arterial chemoreceptors. Moreover, it may also be applied to study other physiological and pathological processes where acute ROS production plays a signaling role. Preparation of CB slices and transduction of roGFP probes by adenoviral infection Timing: 2 h Reagent Final concentration Amount NaCl (MW 58.44) 117 mM 13.675 g KCl,(MW 74.55) 4.5 mM 0.67 g NaHCO 3 (MW 84.01) 23 mM 3.864 g MgCl 2 (1 M) 1 mM 2 mL CaCl 2 (1 M) 2.5 mM 5 mL Glucose (MW 180.16) 5 mM 1.801 g Sucrose (MW 342.3) 5 mM 3.423 g ddH 2 O - up to 2 L Continued Reagent Final concentration Amount Fetal bovine serum 10% 1 mL Insulin (100 U/mL) 84 U/L 8.4 mL Erythropoietin (2000 U/mL) 1.2 U/mL 6 mL ll OPEN ACCESS 4STAR Protocols 2, 100535, June 18, 2021 Protocol MouseCBsaredissectedandsectionedquickly(Ortega-Sa´enz et al., 2018). roGFP probes incorporated in adenoviral vectors are transduced to CB glomus cells by adenoviral infection (Arias- Mayenco et al., 2018). CB slices are then cultured for 48 h before measurement of ROS production by microfluorimetry. 1. Carotid body dissection a. Sacrifice mouse with intraperitoneal administration of a lethal dose of sodium thiopental (120 to 150 mg/kg). b. Quickly dissect carotid bifurcation under a microscope and place it in ice-cooled and modified 0Ca 2+ Tyrode’s solution (Figures 1A and 1B). c. Dissect CB from the carotid bifurcation by removing superior cervical ganglion, adjacent arteries, fat, and connective tissues (Figures 1C–1E). Note: Other anesthetic or ways of sacrifice can also be applied without interfering in the success of the experiment. 2. Carotid body slicing a. Embed CB in 1% (wt/vol) low melting-point agarose in PBS, which has been pre-warmed to 42C, and quickly place the agarose block on ice for solidification (Figures 1F and 1G). Agarose blocks are prepared using a fragment (about 1 cm in length) of 1 mL syringe, with both ends perfectly polished and one end sealed with parafilm (Figures 1F and 1G). b. Remove the solidified agarose block from the syringe fragment, make a smaller agarose cylinder containing the CBs, and fix it to a specimen disc with cyanoacrylate. Place the specimen disc inside the buffer tray of a vibratome (Figure 1H). Fill the tray with cold, modified 0 Ca 2+ Tyrode’s solution. c. Make 150 mm-thick slices using a standard razor blade (Figure 1I). Transfer slices to a plastic container and wash them twice with cold sterile PBS. CRITICAL: The temperature of agarose solution for embedment should be maintained between 40C–42C. A temperature lower than 40C may affect CB embedment and CB sectioning, whereas a temperature above 42C may affect the viability of CB cells. CRITICAL: The speed to section CB embedded in the agarose block needs to be maintained between 0.1 and 0.16 mm/s. CB may detach from agarose block if the speed is too high. Alternatives: Modified 0 Ca 2+ Tyrode’s solution can be replaced with PBS without affecting the quality of CB slices. 3. Brief enzymatic treatment a. Incubate CB sections with enzymatic digestion solution for 5 min at 37Cinawaterbathwith shaking. b. Transfer CB sections to a 35-mm petri dish and wash the sections with cold PBS twice. Note: Enzymatic treatment is performed to partially digest and loosen connective tissues in the surface of CB slices. This treatment seems to help the appearance of glomeruli on the surface of CB slices during culturing. This step is not necessary for rat CB. 4. Culture and transduction of roGFP probes to CB sections by adenoviral infection a. Remove PBS completely and add culture medium, which has been pre-equilibrated in cell culture incubator of 21% O 2 ,5%CO 2 at 37C. ll OPEN ACCESS STAR Protocols 2, 100535, June 18, 2021 5 Protocol CRITICAL: The culture medium should be equilibrated with pH and temperature in the incubator right before use. The correct pH (7.4) of culture medium is important for CB recovery from sectioning and enzymatic treatment, and for survival during the time of culture. Figure 1. Mouse carotid body (CB) dissection and slicing (A) Adult mouse after treatment with lethal dose of sodium thiopental. (B) Dissection of carotid bifurcation with forceps and scissors. Scale bar, 2 mm. (C) Carotid bifurcations before (left) and after (right) removal of superior cervical ganglion (SCG, highlighted on the left bifurcation). CB is highlighted on the right bifurcation. Scale bar, 500 mm. (D) Magnified image of the carotid bifurcation without SCG in C(right). ICA, internal carotid artery; ECA, external carotid artery; CA, carotid artery; CS, carotid sinus. Scale bar, 500 mm. (E) Two CBs dissected from one mouse. Scale bar, 200 mm. (F) Two CBs (arrows) embedded within the agarose block inside the syringe fragment. (G) Agarose block with two embedded CBs (arrows) removed from the syringe. Scale bar, 5 mm. (H) Agarose block (ag) with embedded CBs fixed to a specimen disc inside the buffer tray of a vibratome. The buffer tray is filled with modified 0 Ca 2+ Tyrode’s solution. rb, razor blade. (I) Slices (150-mm thick) of the agarose block (circle) containing a CB section (arrow). The slices are floating in a small plastic tube filled with Modified 0 Ca 2+ Tyrode’s solution. Scale bar, 2 mm. ll OPEN ACCESS 6STAR Protocols 2, 100535, June 18, 2021 Protocol b. Transduce roGFP probes into CB cells by adenoviral infection. roGFP DNA construct has been incorporated previously in adenoviral vector. One ml of adenovirus with a stock concentration of 1–1.2 310 12 viral particles/mL is added to 2 mL of complete culture medium. CRITICAL: The amount of adenovirus necessary for infection may vary from one lot of virus production to another. To maximize transduction efficiency, functional viral titration may be performed, and approximately 400,000 pfu/mL of adenovirus is recommended for each infection. c. Adenovirus with different roGFP constructs are available to target specific subcellular compartments: cytosol, cyto-roGFP (VQAd ro-GFP); mitochondrial intermembrane space (IMS), IMS-roGFP (VQAd CMV GDP-roGFP); and mitochondrial matrix, matrix-roGFP (VQAd CMV mito-roGFP) (Waypa et al., 2010). d. Culture CB sections in a humidified incubator with 5% CO 2 and 21% O 2 at 37Cfor24h.CB slices from each mouse (4–6 slices) are cultured in a 35-mm petri dish. Before putting in the incubator, shake the dish slightly and horizontally to avoid slice overlapping. e. Replace the medium with adenovirus-free culture medium and incubate CB sections for an additional 24 h before microfluorimetric recording of ROS production. Alternatives: If necessary, cells may be incubated with adenovirus-containing culture medium for 48 h until microfluorimetric recording to allow maximum roGFP expression. This may be useful especially when the transduction efficiency is low. CRITICAL: The optimal culturing time of CB slices is 24 to 48 h, which allows the recovery of typical CB glomerulus morphology and response to physiological stimuli, such as hypoxia and hypercapnia. CB slices can be maintained in relatively good conditions for up to 4– 5days(Ferna´ndez-Agu ¨era et al., 2015). However, the number of O 2 responding cells in CB sections decreases with time after reaching a maximum number at about 48 h. It is essential to maintain CB slices healthy, since the response to hypoxia of CB glomus cells canbeeasilylost. Note: When possible, it is recommended to use young adult mice (2–3 months). Although feasible, older mice tend to contain more connective tissue, which may affect the quality of tissue sectioning and enzymatic treatment. Real-time recording of ROS production from CB slices using roGFP probes by microfluorimetry Timing: 1–2 h ROSproductionisrecordedinreal-timeusingamicrofluorimetric setup 48 h after adenoviral infection. As shown in Figure 2A, our microfluorimetric setup consists of a direct microscope equipped with a 603/0.90w objective, a filter wheel, a 150 W xenon lamp, a monochromator, a CCD camera and a computer. The monochromator allows to excite samples at different wavelength. The emitted fluorescence passes through a band-pass filter and is detected by a CCD camera. Monochromator, CCD camera, and image acquisition are controlled by Live Acquisition software. roGFPexpressioninCBsectioncanbeobserved(Figures 2Band2C).ROSproductionindifferent subcellular compartments, such as mitochondrial IMS, mitochondrial matrix, and cytosol, can be measured using roGFP probe targeted to each compartment (Waypa et al., 2010). roGFPs have two fluorescence excitation peaks (400 and 484 nm), which allows ratiometric measurements to determine reversible changes of redox state (Figure 2D). The presence of oxidants causes a disulfide bond formation between cysteine residues in roGFP and an increase in the fluorescent signal at the 400 nm excitation wavelength and a decrease at 480 nm wavelength. In addition, basal level redox ll OPEN ACCESS STAR Protocols 2, 100535, June 18, 2021 7 Protocol state can be determined by in situ calibration of roGFP probes with excess reducing agent (dithiothreitol) to achieve maximum reduction and oxidant (H 2 O 2 ) to achieve maximum oxidation. Effect of hypoxia on ROS production from CB glomus cells can be studied using recording solutions bubbled with gas mixtures containing different O 2 content. 5. Transfer a CB slice to the recording chamber of microfluorimetric setup, which has been filled with pre-warmed recording solution, and maintain a continuous flow of recording solution. Note: The recording solution is pre-warmed to 37C and kept in a water bath of 37Cduring the experiment in order to maintain the recording temperature at 30C–33C. Note: The volume of recording solution in the recording chamber is minimized to z0.2 mL during recording to facilitate fast changes of experimental condition. The flow rate of recording solution is about 4 mL/min. 6. Use a dichroic Di02-R488 (Semrock) and a band-pass filter FF01-520/35 (Semrock) for recording. 7. Select single glomus cell (region of interest, ROI) with strong green fluorescent signal, indicating high level of roGFP expression. Several ROIs can be selected in the same field for simultaneous recording. CRITICAL: To avoid florescent signal bleaching during experiment, it is recommended to decrease exposure time right after ROI selection. Figure 2. Microfluorimetric setup for measurement of reactive oxygen species production using redox-sensitive green fluorescent protein (roGFP) (A) Schematic representation of microfluorimetric setup. See text for details. (B and C) Representative carotid body (CB) slice 48 h after adenoviral infection in bright field (B) and with excitation of 480 nm and emission of 535 nm (C), demonstrating green fluorescent signal due to roGFP expression. A CB glomerulus is highlighted with a white discontinuous line. Scale bar, 10 mm. (D) Representative recording of fluorescent signals excited at 400 nm and 480 nm with an emission at 535 nm and the ratio of fluorescent signals (400/480) of a CB glomus cell. DTT, dithiothreitol. ll OPEN ACCESS 8STAR Protocols 2, 100535, June 18, 2021 Protocol