Feb 2019



An ex vivo Approach to Assess Mitochondrial ROS by Flow Cytometry in AAV-tagged Astrocytes in Adult Mice

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Mitochondrial reactive oxygen species (mROS) are naturally produced signalling molecules extremely relevant for understanding both health- and disease-associated biological processes. The study of mROS in the brain is currently underway to decipher their physiopathological roles and contributions in neurological diseases. Recent advances in this field have highlighted the importance of studying mROS signalling and redox biology at the cellular level. Neurons are especially sensitive to the harmful effects of excess mROS while astrocytic mROS have been shown to play a relevant physiological role in cerebral homeostasis and behaviour. However, given the complexity of the brain, investigating mROS formation in a specific cell-type in adult animals is methodologically challenging. Here we propose an approach to specifically assess mROS abundance in astrocytes that combines i) a targeting strategy based on the use of adeno-associated virus (AAV) vectors expressing the green fluorescent protein (GFP) under an astrocyte (glial fibrillary acidic protein or GFAP) promoter, along with, ii) a robust and widely extended protocol for the measurement of mROS by flow cytometry using commercial probes. The significance of this work is that it allows the selective study of astrocytic mROS abundance by means of easily accessible technology.

Keywords: Reactive Oxygen Species (ROS) (活性氧), Astrocyte (星型胶质细胞), Mitochondria (线粒体), Adeno-associated virus (AAV) vectors (腺相关病毒载体), Oxidative stress (氧化应激), Redox biology (氧化还原生物学), Brain (脑), Neurodegeneration (神经退变)


Oxidative damage is associated with the aetiology of many diseases, including neurodegenerative disorders given that the brain is an exceptionally vulnerable tissue to oxidative stress as well as to age-related alterations (Cobley et al., 2018; Mattson and Arumugam, 2018). Yet there are still important gaps in the understanding of reactive oxygen species (ROS) pathophysiology in the brain. The deleterious effects associated with ROS in situations of redox stress are in contrast to the increasing evidence suggesting that physiological processes are also fine-tuned by ROS (D’Autréaux and Toledano, 2007; Holmström and Finkel, 2014; Hopkins, 2016a and 2016b). In fact, the clinical application of ROS as signalling molecules is still far away from being an effective solution as a translational therapy (Kamat et al., 2008; Carvalho et al., 2017). Thus, the success of antioxidant therapies depends on considering aspects of redox biology such as its pleiotropism or its cellular and subcellular origin (Juránek et al., 2013; Zhang et al., 2016). In vitro studies using brain cells, principally neurons and astrocytes, have been essential for understanding the highly distinctive biology of these cells. In this sense, it has been shown how these brain cells present different molecular specialization, highlighting the importance of crosstalk during neuron-astrocyte coupling which ensures brain bioenergetic and redox homeostasis (Fernandez‑Fernandez et al., 2012; Bolaños, 2016). Regarding the study of ROS in brain cells, there is recent evidence indicating that the levels of mitochondrial reactive oxygen species (mROS) are immensely greater–about one order of magnitude–in astrocytes than in neurons (Lopez-Fabuel et al., 2016). This finding reiterates the relevance of considering the cellular origin of neural ROS as an important new factor in the study of redox biology in the brain.

To address this issue, we have reported the use of a new method to quantify the levels of mROS in adult mice astrocytes ex vivo, independently of neurons (Vicente-Gutierrez et al., 2019). This methodology has allowed us to demonstrate an effective and specific down-modulation of astrocytic mROS in a transgenic mouse model expressing a mitochondrial-tagged form of catalase (mitoCAT or mCAT) (Vicente-Gutierrez et al., 2019). In this particular study, we showed that astrocytic mROS has an impact on neuronal function and survival by regulating bioenergetics and redox metabolism. Thus, by decreasing endogenous levels of astrocytic mROS, we found that astrocytic mROS may modulate glucose utilization and neuronal function in behaving mice (Vicente-Gutierrez et al., 2019). Using the methodology that we herein describe, we postulated that endogenous mROS in astrocytes play a physiological role in the maintenance of brain homeostasis. Furthermore, in this same study (Vicente-Gutierrez et al., 2019), we were able to characterize a novel conditional mCAT mouse that could be useful for testing the implications of mROS in a desired specific tissue type or pathological model.

Since the study of ROS depends not only on concentration but also on its spatiotemporal distribution, real-time imaging of ROS, possibly in vivo, has become necessary in order for scientists to determine which of their biological activities may present a potential for clinical translation. However, this objective is still unachievable owing to current available techniques (Maulucci et al., 2016). Here, we describe in further detail the protocol previously used in Vicente-Gutierrez et al. (2019) to measure mROS abundance, specifically superoxide anion-abundance in astrocytes ex vivo. Although there are different probes commercially available, the most widely used is MitoSOXTM. The MitoSOX Red mitochondrial superoxide indicator enters into mitochondria, where it accumulates in response to the mitochondrial membrane potential (ΔΨm) and becomes oxidized. This probe has been widely used in astrocytes in culture (Sheng et al., 2013; Angelova et al., 2015) and in cultured hippocampal sections (Ishii et al., 2017). MitoSOX fluorescence intensity is commonly assessed by microscope imaging or flow cytometry. As mentioned above, this fluorescent dye requires an active mitochondrial membrane potential to enter into mitochondria. This implies that to measure mROS, it is necessary to use live cells and to determine, in parallel, the ΔΨm in order to confirm that differences in the MitoSOX signal are independent of ΔΨm. However, this fact presents a challenge when working with fixed-cells. For instance, this exclude the use of imaging techniques like immunohistofluorescence in fixed-cells which, in addition, to study individual cellular phenomena required the use of different cell markers. To overcome the cell origin problem, others have used genetically encoded probes to assess redox histology in mouse (Fujikawa et al., 2016). Thus, immunohistochemical strategies still offer a suitable approach for detecting the footprints of redox stress. Nowadays, two-photon laser scanning microscopy has become an interesting tool for studying cellular parameters in awake mice, in vivo. However, this technology is not easily accessible in every laboratory and is limited to studying superficial brain areas (Pérez-Alvarez et al., 2013; Wang et al., 2017). Another drawback to this technique is the large amount of time required for sample preparation and for obtaining replicas. In contrast, the protocol that we will describe is relatively easy, accessible, fast and robust.

The ability of certain adeno-associated virus (AAV) vectors to cross the blood-brain barrier after intravenous injection makes it possible to obtain transgene expression in brain cells. The AAV-PHP.eB capsid is one of the serotypes that is able to efficiently transduce the central nervous system (Chan et al., 2017) and is useful for many applications. For instance, we have used it to express the green fluorescent protein (GFP) under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) short promoter (gfa-ABC1D) (Lee et al., 2008). Astrocytes were specifically tagged in vivo by infecting mice intravenously through the retro-orbital venous sinus with the AAV-PHP.eB-gfa-ABC1D-GFP construct. Then, we obtained a single brain cell suspension and adapted a well-known protocol to measure MitoSOX by flow cytometry. Therefore, this protocol combines the use of accessible technology like flow cytometry with the use of commercial probes. Accordingly, another advantage to this approach is its versatility, since it is also useful for assessing other cell-specific processes in brain function. By using other brain cell-specific promoters to target neurons, microglia or oligodendrocytes, for instance, the same phenomenon could be measured independently or simultaneously in different cell types. Moreover, this protocol allows the same sample to be analysed using different commercially available probes to measure different biological processes. Finally, this tool is greatly useful to study other cellular and subcellular phenomena and can be used at different time points during model lifespan as well as different tissue types. Overall, we believe that this straight-forward protocol for measuring mROS in adult astrocytes is beneficial for studying redox biology in vivo at the cellular level in the brain.

Materials and Reagents

  1. Anaesthesia
    1. Mice C57BL/6J
    2. Sevofluorane (Sevorane®)
    3. Oxygen (O2) Purity: 99.995% (Air Liquide, AlphagazTM)
    4. Nitrous oxide (N2O) (Air Liquide, AlphagazTM, CAS no.: 010024-97-2)

  2. AAVs injection
    1. High level surface disinfectant Rely+OnTM Virkon® (DuPont®)
    2. Barrier and non-filtered pipette Tips (Art tips, Thermo Scientific)
    3. Insulin syringes BD Micro-Fine + Demi 0.3 ml, 30 Gauge, 8 mm (Becton Dickinson, catalog number: 324826 )
    4. AAVs. To target astrocytes in this protocol we use AAV-PHP.eB-gfa-ABC1D-GFP construct
    5. Pluronic F-127 (Sigma-Aldrich, catalog number: P2443-2506 )
    6. Phosphate buffered saline (PBS) (GibcoTM, catalog number: 70011044 )
    7. Parafilm paper (Bemis, PM-996, USA)

  3. Brain single-cell suspension
    1. Microsurgical scissor (Fine Science Tools, catalog number: 91501-09 )
    2. Scalpel blade (N° 24, KRAPE S.A., catalog number: BS EN 27740 )
    3. Fetal bovine serum (FBS) (GibcoTM, catalog number: 10270 )
    4. Bovine serum albumin (Sigma-Aldrich)
    5. DNase I (Roche Diagnostics, catalog number: 10104159001 )
    6. Trypsin (Sigma-Aldrich, catalog number: T4799 )
    7. NaCl (Merck, catalog number: 106404 )
    8. KCl (Merck, catalog number: 104936 )
    9. MgSO4·7H2O (Merck, catalog number: 5886 )
    10. NaHCO3 (Sigma, catalog number: 6329 )
    11. NaH2PO4·2H2O (Merck, catalog number: 106345 )
    12. β-D(+)Glucose (Sigma, catalog number: G5250 )
    13. Phenol red. (Sigma, catalog number: P5530 )
    14. KH2PO4 (Merck, catalog number: 4873 )
    15. HEPES (Sigma, catalog number: H3375 )
    16. CaCl2·2H2O (Merck, catalog number: 102381 )
    17. Earle’s Balanced Salt Solution (EBSS) buffer (see Recipes)
    18. HBSS buffer (see Recipes)
    19. Disaggregation solution (see Recipes)
    20. Resuspension solution (see Recipes)

  4. Mitochondrial ROS measurement
    1. MitoSOXTM Red Mitochondrial Superoxide Indicator (Molecular Probes, Inc., InvitrogenTM, Thermo Fisher, catalog number: M36008 )


  1. Anaesthesia procedure and AAVs handling
    1. Automatic pipettes (Gilson's PIPETMANTM)
    2. Microcentrifuge (Eppendorf, model: Centrifuge 5424 )
    3. Vortex (Scientific Industries, Inc., model: Vortex Genie 2 )
    4. Anaesthesia system composed by a gas distribution column (Hersill H-3, Spain) and a Vaporizer (InterMed Penlons Sigma Delta) (Figure 1)
    5. Class II cabinet equivalent to Telstar Bio II Advance certified according EN-12469-2000

      Figure 1. Anaesthesia system

  2. Mitochondrial ROS measurement
    1. FACSCaliburTM Flow Cytometer (Becton Dickinson, catalog number: 342975 ), equipped with a15 mW argon laser
    2. Centrifuge adapted for Flow cytometry tubes (Eppendorf, model: Centrifuge 5810 R )


  1. BD CellQuestTM Pro version 5.2 (Becton, Dickinson & Company, BD Biosciences)
  2. FlowJo X 10.0.7r2 (FlowJo, LLC, Becton, Dickinson & Company)


  1. Protocol for the intravenous injection of the adeno-associated virus (AAV) through the retro-orbital venous sinus (Figure 2)
    Here, we describe the experimental method designed by Vicente-Gutierrez et al. (2019) using the AAV-PHP.eB-gfa-ABC1D-GFP construct. This protocol is detailed to specifically express a green fluorescent protein (GFP) in the astrocytes of adult mice. However, this approach can be useful to target others cell and tissue types. In that case, using capsids or cell-specific promoters different to AAV-PHP.eB and gfa-ABC1D, respectively, it will be necessary to validate considering aspects like the efficient number of AAVs units, route of administration for the injection or mice age.

    Figure 2. Schematic representation of the protocol to distinguish astrocytes from other neural populations isolated from adult mice by flow cytometry after injection of astroglial AAVs (AAV-PHP.eB-gfa-ABC1D-GFP)

    1. Prepare a unique AAVs suspension for all mice employed.
      1. Particle aggregation is a common problem due to a highly concentrated AAV suspension. This phenomenon could be minimized by increasing the ionic strength of the solution where the AAVs are suspended. To do so, add a non-ionic surfactant like pluronic F-127 to PBS solution (Penaud-Budloo et al., 2018). Use PBS solution containing 0.001% Pluronic F-127 as vehicle solution as well as for the dilutions of AAV suspensions.
      2. Nextstep by step protocol refers to the use of a single AAV construct. However, we recommend using two types of control conditions, namely: 1) mice injected with the vehicle solution (PBS + 0.001% Pluronic F-127) and 2) mice injected with an empty AAV construct (without GFP) prepared in the same vehicle solution at the desired viral concentration. Both control mice will help to eliminate any possible unwanted responses after infection, as well as to easily distinguish GFP expression in the AAV-GFP-injected mice.
      3. All manipulation should be done within a protective class II laminar flow cabinet. Products in contact with the AAVs must be decontaminated with Rely+OnTM Virkon®.
      1. Calculate the number of mice to be injected on the same day and take the final volume of the AAV suspension needed for all of them. Each adult mouse is injected with 50 µl of AAV suspension. Hence, the total volume of AAV suspension is calculated as VT = 50 µl x the number of injected mice.
      2. Prepare 5 x 1010 viral genomes of AAV-PHP.eB-gfa-ABC1D-GFP per adult mouse in vehicle solution (PBS + 0.001% Pluronic F-127). This concentration is based on our previous experience, although it is advisable to check the expression of the protein of interest in the target tissue for each AAV serotype and/or which each promoter. It should be taken into account that the volume of AAVs suspension (VAAV) may vary according to the dilution of the stock of the concentrated AAV suspension.
      3. Add the vehicle solution (PBS + 0.001% Pluronic F-127) to the to the AAVs suspension (VAAV) in a microcentrifuge tube using a volume equal to (VT-VAAV) to obtain a volume that will permit 50 µl to be injected into each adult mouse. This ensures the desired working dilution of 5 x 1010 viral genomes per 50 µl of AAVs suspension.
      4. Vortex the microcentrifuge tube with the vehicle+AAVs suspension mixture at a working dilution for 10-15 s.
      5. Centrifuge the tube for 20 s at full speed (~21,000 x g) in a microcentrifuge to recover the total amount of the prepared AAVs suspension.
      6. Prepare individual single-use 30 Gauge syringes containing 50 µl of the AAVs suspension for each mouse.
        1. Use an automatic pipette to ensure accuracy when loading each syringe.
        2. Homogenize by repetitive pipetting (~9 times) before loading each syringe with the 50 µl aliquot.
        Note: Avoid the formation of bubbles when loading the syringes. An easy way to accurately load the syringes without creating bubbles is to prepare a 50 µl drop on a piece of parafilm after homogenization. Then, load each syringe from the parafilm with the bevel down.
    2. Mice should be briefly anaesthetized with sevofluorane at 6% for initiation followed by ~3% for maintenance in air with supplemented O2 and NO2 (0.4 and 0.8 L/min, respectively) with a gas distribution column and a vaporizer.
      Note: Sevoflurane should be administered by a professional trained in the administration of anaesthesia. Respiration should be supervised during sevoflurane administration, and the dose should be reduced upon signs of muscle rigidity.
    3. Inject a 50 µl aliquot into each anaesthetized animal through the retro-orbital venous sinus and return each one to its home cage (see Yardeni et al., 2011 for specific advice and considerations of this intravenous route of administration).
      Note: This route of administration requires specific training. To avoid forcing or making pressure trough eye orbit, the needle is carefully introduced, bevel down, at an angle of approximately 30°, into the medial canthus. Often a small drop of blood is observed at the injection site after administration, which can be cleaned using medical gauze. The total duration of the procedure per mouse is less than 10 min, including injection, anaesthesia and recovery from the effects of the narcotic.
    4. Wait three weeks post-injection after the AAV construct has time to be expressed. Then, the astrocyte population of interest can be selected using fluorescent flow cytometry.
      Note: Any AAV-containing waste, including bedding and mice faeces, should be collected and placed into biohazard bags during the first week. The waste should then be autoclaved to inactive the AAV. After injection, all mice should be kept in quarantine for at least 3 weeks to avoid possible immune responses, as well as to allow for transgene expression.

  2. Protocol for measuring the mitochondrial reactive oxygen species (mROS) in brain single cell suspensions
    This should be performed three weeks after infection with the AAV-PHP.eB-gfa-ABC1D-GFP construct expressing GFP in the astrocytes of adult mice. Follow the steps of the protocol described below for extracting the brain and preparing single cell suspension for measuring mROS using the fluorescent commercial probe MitoSOX (Lopez-Fabuel et al., 2016; Vicente-Gutierrez et al., 2019).

    Protocol for obtaining single cell suspension of brain tissue:
    1. Sacrifice the mice using cervical dislocation avoiding the use of any asphyxiation method that could interfere with ROS production and bias the subsequent ROS measurement.
    2. Extract the brain and, if necessary, isolate the brain regions of interest.
    3. At room temperature (RT), rinse the brain tissue in PBS and immerse it in 1 ml of EBSS solution (Recipe 1).
    4. Prepare a single-cell suspension from the brain tissue following an enzymatic and mechanical disaggregation procedure (originally described by Almeida and Medina, 1998):
      1. Aspirate the total amount of EBSS solution.
      2. Cut the brain or brain section using microsurgical scissors or a scalpel blade to obtain ~3 mm2 pieces.
      3. Add 500 µl of the dissociation solution (Recipe 2) for a whole brain or scale the volume accordingly for smaller pieces.
      4. Incubate at 37 °C for 10 min with shaking to allow a mild enzymatic dissociation to occur.
      5. Neutralize trypsinization with 10% fetal bovine serum (FBS) to halt the enzymatic dissociation.
      6. Centrifuge the tissue (5 min, 500 x g) and discard the supernatant.
      7. Add 1 ml of resuspension solution (Recipe 3) and resuspend the pellet using a Pasteur pipette and do a repetitive pipetting (10-15 times) to get a smooth mechanical disaggregation.
      8. Leave the suspension to settle for 5 min and then transfer the supernatant (containing single cells) to a new tube.
      9. Add an additional 1 ml of resuspension solution to the remaining pellet and repeat the homogenising process (Steps B4g-B4h) to obtain a final volume of ~2 ml of a single-brain cell suspension (repeat once more if necessary).

    Measurement of Mitochondrial ROS abundance:
    1. Split each sample into 4 aliquots: one will be used as a negative control for MitoSOX staining and the other three as test sample technical replicates. The negative control should be resuspended in an appropriate volume (1 ml per 106 brain cells) of HBSS solution (Recipe 4). The following steps refer to the treatment of the test samples.
    2. Incubate the adult brain cell suspension (use at least 3 technical replicates per sample) together with the fluorescent probe MitoSOX.
      1. Prepare MitoSOX working solution; 3 µM MitoSOX reagent in HBSS buffer (Recipe 4).
      2. Centrifuge the brain cell suspension to obtain a pellet (5 min, 500 x g).
      3. Resuspend the pellet by gently and manually shaking the tubes after adding 1 ml of MitoSOX working solution at approximately 106 brain cells.
        Note: Count cells to adapt the final volume of MitoSOX solution to the total number of cells.
      4. Incubate the cells for 30 min at 37 °C protected from light, according to the manufacture’s protocol.
      5. After incubation, wash cells once with 2 ml of warm HBSS buffer.
      6. Centrifuge the brain-cell suspension to remove any excess MitoSOX (5 min, 500 x g).
      7. Resuspend in HBSS buffer using an appropriate volume to obtain a single-cell suspension to be analysed in the flow cytometer.
        Note: An appropriate volume per replicate derived from a whole brain is 500 µl, scale the volume accordingly for smaller areas. This volume allows a final dilution to asses less than 300 events/second tolerable to FACScalibur, considering an ‘event’ a singular cell.
      8. For flow cytometry, use approximately 510 nm excitation and a fluorescence channel able to detect the 580 nm emission of oxidized MitoSOX reagent (FL-3 in BD FACSCaliburTM flow cytometer).
    3. Flow cytometry:
      Note: Flow cytometry requires previous training to set compensation of signal fluorescence and to adapt the parameters of the flow cytometer to brain cells size and complexity.
      1. Use tubes containing the control samples to calibrate the desired voltage adjustments for each channel used to detect single or combined stains. The compensation of each channel avoids the interference of fluorescence signals in different channels. The control samples used for the purpose of calibration in this protocol are:
        Non-stained cells. Cells without any stain (no MitoSOX) derived from vehicle-injected mice (no GFP).
        Cells exclusively positive for MitoSOX channel (FL-3 in BD FACSCaliburTM flow cytometer). Cells derived from vehicle-injected mice incubated with MitoSOX.
        Cells exclusively positive for GFP channel (FL-1 in BD FACSCaliburTM flow cytometer).
        Cells derived from any mice injected with AAVs-GFP (AAV-PHP.eB-gfa-ABC1D-GFP) without incubation with MitoSOX.
        Note: As mentioned in Steps B5, it is advisable to split one replicate per sample (mouse/brain area) to obtain a non-stained negative control sample to eliminate unwanted measurements in the MitoSOX channel.
      2. Finally, test samples should be assessed for both previously calibrated channels:
        1. First, set the voltage in forward (FSC: cell size) and side scatter (SSC: cell complexity) to centre the population and properly visualize the sample in the dot plot XY graph.
        2. Calibrate and compensate channels using the control samples (Review StepB7a above).
        3. Once calibrated, pass each tube using the established parameters and record at least ~100,000-200,000 events (this number may vary depending on the expected percentage of GFP positive population).

Data analysis

Use FlowJo or similar software to quantify the intensity of MitoSOX signal in the brain samples. First, gate on the cells excluding debris or doublets events looking at forward (size) and side (complexity) scatter scales. Then, measure the thresholds for GFP and MitoSOX signals with negative control samples used to perform standard compensation (Figure 3). Once these limits are established, determine the mean of the fluorescent intensity values for the events included in the gate of interest.
  It is advisable to use at least three technical replicates for each sample to obtain a robust mean for each biological replicate. Results are expressed as arbitrary units of signal intensities.
  Figure 3 represents the workflow used in the flow cytometry analysis, which served to show that mice expressing a mitochondrial-tagged isoform of catalase (mCAT) in astrocytes present lower levels of astrocytic mROS (see Vicente-Gutierrez et al., 2019).

Figure 3. Flow cytometry workflow for brain cells experiments to measure mitochondrial ROS (mROS) (using MitoSOX) exclusively in astrocytes expressing GFP after the infection of mice with AAV-PHP.eB-gfa-ABC1D-GFP (original data is contained in Vicente-Gutierrez et al., 2019).


Take into account that some of these solutions are commercially available.

  1. Earle’s Balanced Salt Solution (EBSS) buffer
    116 mM NaCl
    5.4 mM KCl
    1.5 mM MgSO4
    26 mM NaHCO3
    1.01 mM NaH2PO4·2H2O
    4 mM glucose
    Phenol red 10 mg/L pH 7.2
  2. Dissociation/Disaggregation solution
    EBSS solution supplemented with bovine serum albumin 0.3% (p/v) (Sigma-Aldrich)
    DNase I 20 μg/ml (Roche)
    Trypsin 0.025% p/v (Sigma-Aldrich) (Almeida and Medina, 1998)
  3. Resuspension solution
    EBSS solution supplemented with supplemented with bovine serum albumin 0.3% (p/v) (Sigma-Aldrich)
    DNase I 20 μg/ml (Roche) (Almeida and Medina, 1998)
  4. HBSS buffer
    134.2 mM NaCl
    5.26 mM KCl
    0.43 mM KH2PO4
    4.09 mM NaHCO3
    0.33 mM Na2HPO4·2H2O
    5.44 mM glucose
    20 mM HEPES
    20 mM CaCl2·2H2O
    pH 7.4


We acknowledge the technical assistance and mice care carried out by M. Carabias-Carrasco, L. Martin, E. Prieto-Garcia and M. Resch from the University of Salamanca as well as the laboratory of G. Bonvento at the Molecular Imaging Center (MIRCen), CNRS UMR 9199, Université Paris, France for providing the AAVs and helpful advice. We would also like to thank Nicoló Bonora and Daniel Jimenez-Blasco who participated in the experimental work involving this procedure.
  This work was funded by MINECO (SAF2016-78114-R), Instituto de Salud Carlos III (CB16/10/00282), NIH/NIDA (1R21DA037678-01), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA, Fundación Ramón Areces and Junta de Castilla y León (Escalera de Excelencia CLU-2017-03). This protocol was originally described by Vicente-Gutierrez et al. (2019).

Competing interests

The authors declare no competing interests.


All animal procedures we performed according to the European Union Directive 86/609/EEC and Recommendation 2007/526/EC, regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish legislation under the directive RD1201/2005. All protocols were approved by the Bioethics Committee of the University of Salamanca.


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[摘要] 线粒体活性氧(mROS )是天然产生的信号分子,与理解健康和疾病相关的生物过程极为相关。目前正在研究大脑中的mROS ,以了解其在神经疾病中的生理病理作用和贡献。该领域的最新进展凸显了在细胞水平研究mROS 信号传导和氧化还原生物学的重要性。神经元对过量的mROS 的有害作用特别敏感,而星形细胞的mROS对 已经显示出在脑稳态和行为中起相关的生理作用。但是,鉴于大脑的复杂性,研究成年动物中特定细胞类型中mROS的形成在方法上具有挑战性。在这里,我们提出了一种方法来具体评估星形胶质细胞中的mROS 丰度,该方法结合了i )一种靶向策略,该策略基于在星形胶质细胞(胶质纤维酸性蛋白或GFAP)下表达绿色荧光蛋白(GFP)的腺相关病毒(AAV)载体的使用)启动子,以及ii)使用商业探针通过流式细胞术测量mROS 的强大且广泛扩展的方案。这项工作的意义在于,它允许通过易于使用的技术来选择性研究星形细胞mROS的丰度。

[背景 ] 氧化损伤与许多疾病的病因有关,包括神经退行性疾病,因为大脑是氧化应激以及与年龄相关的变化异常脆弱的组织(Cobley 等,2018; Mattson和Arumugam ,2018)。然而,在大脑中对活性氧(ROS)病理生理学的理解上仍然存在重要的空白。与氧化还原应激下活性氧相关的有害作用与越来越多的证据表明活性氧也可以对生理过程进行微调形成对比(D'Autréaux和Toledano ,2007;Holmström和Finkel ,2014; Hopkins ,2016 a和2016)b )。实际上,ROS作为信号传导分子的临床应用距离作为转化疗法的有效解决方案还很遥远(Kamat 等,2008; Carvalho 等,2017)。因此,抗氧化治疗的成功依赖于考虑的氧化还原生物学的方面,诸如其pleiotro PISM 或其细胞和亚细胞来源(Juránek 等人,2013;张等人,2016) 。使用脑细胞(主要是神经元和星形胶质细胞)进行的体外研究对于理解这些细胞的高度独特生物学至关重要。从这个意义上讲,已经证明了这些脑细胞如何呈现出不同的分子专一性,突显了神经元-星形细胞偶联过程中串扰的重要性,这确保了大脑的生物能和氧化还原稳态(Fernandez - Fernandez 等,2012 ;Bolaños ,2016 )。关于脑细胞中ROS的研究,最近的证据表明线粒体活性氧(mROS )的含量要高得多- 大约一个数量级。 –星形胶质细胞比神经元(Lopez-Fabuel 等,2016)。这一发现重申了将神经ROS的细胞起源视为研究脑中氧化还原生物学的重要新因素的相关性。

为了解决这个问题,我们已经报道了一种新方法的使用,该方法可以独立于神经元来定量定量离体成年小鼠星形胶质细胞中mROS的水平(Vicente-Gutierrez et al。,2019)。这种方法学使我们能够在表达线粒体标记过氧化氢酶(mitoCAT 或mCAT )的转基因小鼠模型中证明星形细胞mROS的有效和特异性下调(Vicente-Gutierrez 等,2019)。在这项特定的研究中,我们表明星形细胞mROS 通过调节生物能和氧化还原代谢对神经元功能和存活产生影响。因此,通过降低星形细胞mROS的末端遗传水平,我们发现星形细胞mROS 可以调节行为小鼠的葡萄糖利用和神经元功能(Vicente-Gutierrez 等人,2019)。使用我们在此描述的方法,我们假设星形胶质细胞中的内源性mROS 在维持脑稳态中起着生理作用。此外,在同一项研究中(Vicente-Gutierrez 等,2019),我们能够表征新型条件mCAT 小鼠,该小鼠可用于测试mROS 在所需特定组织类型或病理模型中的意义。

由于对ROS的研究不仅取决于浓度,还取决于其时空分布,所以对ROS(可能在体内)的实时成像已成为必要的,以便科学家确定其生物学活性中哪些可能具有临床翻译潜力。但是,由于当前可用的技术,该目标仍然无法实现(Maulucci 等,2016)。在这里,我们将更详细地描述先前在Vicente-Gutierrez 等人中使用的协议。(2019)来测量活性氧代谢丰度,SP ecifically超氧阴离子丰度在星形胶质细胞的离体。尽管市售有不同的探针,但使用最广泛的是MitoSOX TM 。所述MitoSOX 红色线粒体超指示器进入线粒体,在那里它响应于累积的线粒体膜电位(ΔΨ 米)和被氧化。该探针已被广泛用于培养中的星形胶质细胞(Sheng 等,2013 ;Angelova 等,2015 )和培养的海马切片(Ishii 等,2017 )。MitoSOX 荧光强度通常通过显微镜成像或流式细胞术评估。如上所述,该荧光染料需要活性的线粒体膜电位才能进入线粒体。这意味着测量活性氧代谢,有必要使用活细胞,并确定,并行地,ΔΨ 米,以便确认的是,在不同MitoSOX 信号是独立的ΔΨ 米。但是,在使用固定单元时,这一事实提出了挑战。例如,这排除了在固定细胞中使用像免疫组织荧光这样的成像技术,此外,为了研究单个细胞现象还需要使用不同的细胞标记物。为了克服细胞起源问题,其他人已经使用遗传编码的探针来评估小鼠中的氧化还原组织学(Fujikawa 等人,2016)。因此,免疫组织化学策略仍然为检测氧化还原应激的足迹提供了一种合适的方法。如今,双光子激光扫描显微镜已成为研究体内清醒小鼠细胞参数的有趣工具。然而,这种技术并非在每个实验室都容易获得,并且仅限于研究浅表脑区域(Pérez-Alvarez 等,2013; Wang 等,2017)。该技术的另一个缺点是样品制备和获得复制品需要大量时间。相反,我们将描述的协议相对容易,可访问,快速且健壮。

静脉注射后某些腺相关病毒(AAV)载体穿过血脑屏障的能力使得在脑细胞中获得转基因表达成为可能。AAV- PHP.eB 衣壳是能够有效转导中枢神经系统的血清型之一(Chan 等人,2017),可用于许多应用。例如,我们已经使用它在星形胶质细胞特异性神经胶质原纤维酸性蛋白(GFAP)短启动子(gfa-ABC 1 D)的控制下表达绿色荧光蛋白(GFP )(Lee 等,2008)。通过用AAV-PHP.eB-gfa-ABC 1 D-GFP构建体通过眼眶后静脉窦静脉内感染小鼠,在体内特异性标记星形胶质细胞。然后,我们获得了单个脑细胞悬浮液,并采用了众所周知的方案以通过流式细胞仪测量MitoSOX 。因此,该协议将可访问技术(如流式细胞仪)的使用与商业探针的使用相结合。因此,该方法的另一个优点是它的多功能性,因为它也可用于评估大脑功能中的其他细胞特异性过程。例如,通过使用其他脑细胞特异性启动子靶向神经元,小胶质细胞或少突胶质细胞,可以在不同细胞类型中独立或同时测量相同现象。而且,该方案允许使用不同的市售探针测量不同的生物过程来分析同一样品。最后,该工具对于研究其他细胞和亚细胞现象非常有用,并且可以在模型寿命以及不同组织类型的不同时间点使用。总的来说,我们认为这种直接测量成人星形胶质细胞mROS的方案对于在脑内细胞水平研究体内氧化还原生物学是有益的。

关键字:活性氧, 星型胶质细胞, 线粒体, 腺相关病毒载体, 氧化应激, 氧化还原生物学, 脑, 神经退变



米冰C57BL / 6J
Sevofluorane (Sevorane ® )
氧气(O 2 )纯度:99 。995%(液化空气,Alph agaz TM )
一氧化二氮(N 2 O)(液化空气,Alphagaz TM ,CAS号:0 10024-97-2 )

高电平表面消毒剂依靠+论TM 卫可® (杜邦® )
Barrie r和未过滤的移液器吸头(艺术吸头,Thermo Scientific)
胰岛素注射器BD Micro-Fine + Demi 0 。3毫升,30规格,8毫米(Becton Dickinson ,目录号:324826)
AAV。为了在该方案中靶向星形胶质细胞,我们使用AAV-PHP.eB-gfa-ABC 1 D-GFP构建体
Pluronic F-127(Sigma-Aldrich,目录号:P2443-2506)
磷酸盐缓冲盐水(PBS)(Gibco TM ,目录号:70011044)

手术刀刀片(N ° 24,KRAPE SA,目录号:BS EN 27740)
˚F 等人牛血清(FBS) (Gibco公司TM ,目录号:10270)
DNase I(Roche Diagnostics,目录号:10104159001)
胰蛋白酶(Sigma-Aldrich ,目录号:T4799)
NaCl (Merck,目录号:106404)
KCl (Merck,目录号:104936)
MgSO 4 · 7H 2 O(默克,目录号:5886)
NaHCO 3 (Sigma,目录号:6329)
NaH 2 PO 4 ·2H 2 O (默克,目录号:106345)
β- D(+)葡萄糖(Sigma,目录号:G 5250)
酚红。(西格玛,目录号:P 5530)
KH 2 PO 4 (默克,目录号:4873)
HEPES(Sigma,目录号:H 3375)
CaCl 2 ·2H 2 O(默克,目录号:102381)
Disagg reging 解决方案(请参阅食谱)
RESU spension溶液(见食谱)

MitoSOX TM 红色线粒体超氧化物指示剂(Molecular Probes,Inc.,Invitrogen TM ,Thermo F 垫圈,目录号:M36008)



自动移液器(Gils on的PIPETMAN TM )
微量离心机(Eppendorf,型号:Centrifuge 5424)
Vortex(科学工业公司,型号:Vortex Genie 2)
由气体分配塔(Hersill H-3,西班牙)和蒸发器(InterMed Penlons Sigma Delta)组成的麻醉系统(图1)
相当于Telstar Bio II Advance的II类机柜已通过EN-12469-2000认证

D:\ Reformatting \ 2020-1-6 \ 1902540--1311 JuanBolaños745122 \ Figs jpg \ Fig1.jpg

图1 。麻醉系统


FACSCalibur TM 流式细胞仪(Becton Dickinso n ,目录号:342975),配备了15 mW的氩激光器
适用于流式细胞仪试管的离心机(Eppendorf,型号:C entrifuge 5810 R)



BD CellQuest TM Pro 5.2版(Becton,Dickinson &Company和BD Biosciences)
FlowJo X 10.0.7r2(FlowJo ,LL C,Becton,Dickinson&Company)



在这里,我们描述了Vicente-Gutierrez 等人设计的实验方法。(2019)使用AAV-PHP.eB-gfa-ABC 1 D-GFP构建体。该协议的详细说明是在成年小鼠的星形胶质细胞中特异性表达绿色荧光蛋白(GFP)。但是,这种方法可以是目标别人细胞和组织类型非常有用。在那种情况下,分别使用分别不同于AAV - PHP.eB 和gfa-ABC 1 D的衣壳或细胞特异性启动子,有必要进行验证,例如AAVs单位的有效数量,注射途径或给药途径等老鼠的年龄。


D:\ Reformatting \ 2020-1-6 \ 1902540--1311 JuanBolaños745122 \ Figs jpg \ Fig2.jpg

图2 。注射星形胶质AAV(AAV-PHP.eB-gfa-ABC 1 D-GFP)后通过流式细胞术区分星形胶质细胞与其他成年小鼠神经细胞的方法


不Ë 小号:

由于高度浓缩的AAV悬浮液,粒子聚集是一个常见的问题。通过增加悬浮AAV的溶液的离子强度,可以最大程度地减少这种现象。为此,向PBS溶液中添加非离子表面活性剂(如普朗尼克F-127)(Penaud-Budloo等人,2018)。使用含有0.001%Pluronic F-127的PBS溶液作为载体溶液以及AAV悬浮液的稀释液。
接着步骤一步协议指的是使用一个单一的AAV构建体。但是,我们建议使用两种类型的对照条件,即amely:1)注射了媒介溶液(PBS + 0.001%Pluronic F-127)的小鼠,以及2)注射了在同一溶液中制备的空AAV构建体(无GFP)的小鼠所需病毒浓度的载体溶液。两只对照小鼠都将有助于消除感染后任何可能的有害反应,并易于区分注射AAV-GFP的小鼠中的GFP表达。
所有操作均应在II级防护层流柜内进行。与自动增值服务接触的产品必须进行净化依靠+ O ň TM 卫可® 。
计算同一天要注射的小鼠数量,并计算所有小鼠所需的AAV悬浮液的最终体积。给每只成年小鼠注射50 µl AAV悬浮液。因此,AAV悬浮液的总体积计算为V T = 50 µl x注射小鼠的数量。
每只成年小鼠在媒介溶液(PBS + 0.001%Pluronic F-127 )中制备5 x 10 10 AAV-PHP.eB-gfa-ABC 1 D-GFP 病毒基因组。该浓度基于我们以前的经验,尽管建议检查每种AAV血清型和/或每种启动子在靶组织中目的蛋白的表达。应考虑到AAV 悬浮液的体积(V AAV )可能会根据浓缩AAV悬浮液的库存稀释度而变化。
将载液(PBS + 0.001%Pluronic F-127 )加入微量离心管的AAVs悬浮液(V AAV )中,使其体积等于(V T - V AAV ),得到的体积为50μl。注入每只成年小鼠。这确保了5×10个所需的工作稀释度10 VI 每50μl的AAV悬浮液的RAL基因组。
用工作稀释液中的媒介物+ AAVs 悬浮液涡旋微量离心管10-15 s。
离心反应管全速20秒(〜21 ,000 X 克在微量)以回收所制备的AAV悬浮液的总量。
为每只小鼠准备单独的一次性30针注射器,其中装有50 µl AAVs悬液。
通过重复吸液(约9次)匀浆,然后将50 µl等分试样装入每个注射器。 
注意:装入注射器时,避免形成气泡。准确加载注射器而不会产生气泡的一种简单方法是在均质后在一块封口膜上准备50 µl的液滴。然后,将斜角向下的每个注射器从封口膜装入。

小鼠应先用6%的七氟烷麻醉,然后用气体分配塔和气化器在补充有O 2 和NO 2 (分别为0.4 L 和0.8 L / min)的空气中以3%的浓度进行麻醉。

通过眼眶后静脉窦向每只麻醉动物中注入50 µl等分试样,并将每只动物放回其家笼中(有关这种静脉内给药途径的具体建议和注意事项,请参见Yardeni 等,2011)。



测量脑单细胞悬浮液中线粒体活性氧(mROS )的协议
应在成年小鼠的星形胶质细胞中感染表达GFP 的AAV-PHP.eB-gfa-ABC 1 D-GFP构建体后三周进行。遵循下面描述的方案的步骤,使用荧光商业探针MitoSOX 提取大脑并准备用于测量mROS的单细胞悬液(Lopez-Fabuel 等人,2016; Vicente-Gutierrez 等人,2019)。



在室温(RT)下,用PBS冲洗大脑组织并将其浸入1 ml EBSS溶液中(配方1 )。
用显微外科手术剪刀或手术刀刀片切开大脑或脑部切片,获得〜3 mm 2 块。   
为整个大脑添加500μl解离溶液(配方2 ),或为小块相应地缩放体积。
在37孵育 ℃下 10分钟,在摇动以允许发生温和的酶解离。
离心组织(5分钟,500 x g )并丢弃上清液。
加入1 ml的重悬溶液(第3条),然后用巴斯德移液器重悬沉淀,重复进行移液(10-15次),以实现平滑的机械分解。
向剩余的沉淀物中再添加1 ml重悬溶液,并重复均质过程(S teps B4g-B4h),以获得最终体积约2 ml的单脑细胞悬液(如有必要,重复一次)。


将每个样品分成4等份:一个将用作MitoSOX 染色的阴性对照,另外三个将作为测试样品的技术重复样品。阴性对照应重悬于适当体积(每10 6个脑细胞1毫升)的HBSS溶液中(配方4 )。以下步骤涉及测试样品的处理。
与荧光探针MitoSOX 一起孵育成人脑细胞悬浮液(每个样品至少使用3个技术重复样品)。
准备MitoSOX 工作溶液;HBSS缓冲液中的3 µM MitoSOX 试剂(配方4 )。
离心脑细胞悬浮液以获得沉淀(5分钟,500 x g )。
在大约10 6个脑细胞中加入1 ml MitoSOX 工作溶液后,通过轻轻并手动摇动试管来重悬沉淀。
注意:对细胞进行计数,以使MitoSOX 溶液的最终体积适应细胞总数。

孵育后,用2 ml温暖的HBSS缓冲液洗涤细胞一次。
离心脑细胞悬液以去除任何多余的MitoSOX (5分钟,500 x g )。
注意:从整个大脑中获得的每个重复样本的适当体积为500 µl,并针对较小的区域相应地缩放体积。考虑到“事件”是单个细胞,该体积允许最终稀释以评估FACScalibur 容许的小于300 事件/秒。

对于流式细胞仪,请使用大约510 nm激发光和一个荧光通道,该通道能够检测580 nm发射的氧化MitoSOX 试剂(BD FACSCalibur TM 流式细胞仪中的FL-3 )。

非染色细胞。Ç ELLS无任何染色(无MitoSOX 从车辆注射的小鼠(无GFP)导出)。

仅对MitoSOX 通道呈阳性的细胞(BD FACSCalibur TM 流式细胞仪中的FL-3 )。用MitoSOX 培养的注射有媒介物的小鼠的细胞。

仅对GFP通道呈阳性的细胞(BD FACSCalibur TM 流式细胞仪中的FL-1 )。
从任何小鼠衍生的细胞注射的AAV-GFP(AAV-PHP.eB-GFA-ABC 1 d-GFP)而不与孵育MitoSOX 。

注意:正如所提到小号TEPS B5,最好是分割每采样1个复制(小鼠/脑区域),以获得非染色阴性对照样品,以消除不需要的测量MitoSOX 信道。




使用FlowJo 或类似软件来定量脑样本中MitoSOX 信号的强度。首先,观察前向(尺寸)和侧面(复杂度)散射标度,排除碎片或双峰事件的单元上的门。然后,使用用于执行标准补偿的阴性对照样品测量GFP和MitoSOX 信号的阈值(图3)。一旦建立了这些限制,就可以确定感兴趣的门中包括的事件的荧光强度值的平均值。


  图3代表流式细胞仪分析中使用的工作流程,该流程显示了星形胶质细胞中表达线粒体标签过氧化氢酶(mCAT )的亚型的小鼠星形胶质细胞mROS 含量较低(见Vicente-Gutierrez 等,2019)。


D:\ Reformatting \ 2020-1-6 \ 1902540--1311 JuanBolaños745122 \ Figs jpg \ Fig3.jpg

图3 。流式细胞仪工作流用于脑细胞实验,以测量AAV-PHP.eB-gfa-ABC 1 D-GFP 感染小鼠后仅表达GFP的星形胶质细胞中的线粒体ROS(mROS )(使用MitoSOX )(原始数据包含在Vicente- Gutierrez et al。,2019)。





厄尔' 小号巴兰土木工程署盐溶液(EBSS)缓冲液


1.5 mM硫酸镁4


1.01毫米NaH 2 PO 4· 2H 2 O

4 mM葡萄糖

P 苯酚的制备红10毫克/ 大号pH 7.2的

解离/ 分解解决方案
EBSS溶液补充了0.3%(p / v)的牛血清白蛋白(Sigma-Aldrich)

DNase I 20μg / ml(罗氏(Roche))

胰蛋白酶0.025%p / v(Sigma-Aldrich)(Almeida和Medina ,1998)

EBSS溶液补充了0.3%(p / v)的牛血清白蛋白(Sigma-Aldrich)

DNase I 20μg / ml(罗氏(Rome ))(Almeida和Medina ,1998)


5.26 毫米氯化钾

0.43毫米KH 2 PO 4

4.09 mM碳酸氢钠3

0.33 mM Na 2 HPO 4 ·2H 2 O

5.44 mM葡萄糖


20毫米CaCl 2 ·2H 2 O





我们感谢由萨拉曼卡大学的M. Carabias -Carrasco,L。Martin,E。Prieto-Garcia和M. Resch以及分子成像中心的G. Bonvento 实验室提供的技术援助和小鼠护理。(MIRCen ),法国巴黎大学CNRS UMR 9199 提供了AAV和有用的建议。我们还要感谢NICOLO 博诺拉和丹尼尔Jimenez- 布拉斯科谁参与了涉及此过程中的试点工作。

  这项工作是由MINECO(SAF2016-78114-R),萨洛德·卡洛斯三世研究所(CB16 / 10/00282),NIH / NIDA(1R21DA037678-01),Ayudas EquiposInvestigaciónBiomedicina 2017FundaciónBBVA,FundaciónRamónAreces和Junta de资助的卡斯蒂利亚·莱昂(Escalera de Excelencia CLU-2017-03)。该协议最初由Vicente-Gutierrez 等人描述。(2019)。








我们按照欧盟指令86/609 / EEC和建议2007/526 / EC所执行的所有动物程序,有关用于实验和其他科学目的的动物的保护,在西班牙立法中根据指令RD1201 / 2005强制执行。所有方案均已获得萨拉曼卡大学生物伦理委员会的批准。




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引用:Vicente-Gutierrez, C. and Bolaños, J. P. (2020). An ex vivo Approach to Assess Mitochondrial ROS by Flow Cytometry in AAV-tagged Astrocytes in Adult Mice. Bio-protocol 10(6): e3550. DOI: 10.21769/BioProtoc.3550.

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