Visualising Neutrophil Actin Dynamics in Zebrafish in Response to Laser Wounding Using Two-Photon Microscopy
利用双光子显微镜观察激光损伤下斑马鱼中性粒细胞的肌动蛋白动态
发布: 2024年06月05日第14卷第11期 DOI: 10.21769/BioProtoc.4997 浏览次数: 2034
评审: Alberto RissoneIvonne SehringAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2022
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Abstract
Cells need to migrate along gradients of chemicals (chemotaxis) in the course of development, wound healing, or immune responses. Neutrophils are prototypical migratory cells that are rapidly recruited to injured or infected tissues from the bloodstream. Their chemotaxis to these inflammatory sites involves changes in cytoskeletal dynamics in response to gradients of chemicals produced therein. Neutrophil chemotaxis has been largely studied in vitro; few assays have been developed to monitor gradient responses in complex living tissues. Here, we describe a laser-wound assay to generate focal injury in zebrafish larvae and monitor changes in behaviour and cytoskeletal dynamics. The first step is to cross adult fish and collect and rear embryos expressing a relevant fluorescent reporter (for example, Lifeact-mRuby, which labels dynamic actin) to an early larval stage. Subsequently, larvae are mounted and prepared for live imaging and wounding under a two-photon microscope. Finally, the resulting data are processed and used for cell segmentation and quantification of actin dynamics. Altogether, this assay allows the visualisation of cellular dynamics in response to acute injury at high resolution and can be combined with other manipulations, such as genetic or chemical perturbations.
Key features
• This protocol is designed to trigger laser wound in zebrafish larvae using two-photon intravital microscopy.
• The ability to wound while imaging makes it possible to monitor the behaviour and actin changes of the cells immediately after gradient exposure.
• The protocol requires a two-photon microscope for best results. Compared with one-photon laser wounding, the injury is more precise and has better tissue penetration.
• The focal nature of the wounds is suitable for studies of neutrophil swarming/aggregation and can be further adapted to infectious settings.
Keywords: Cell migration (细胞迁移)
Neutrophil (中性粒细胞)
Two-photon imaging (双光子成像)
Laser wounding (激光损伤)
Actin dynamics (肌动蛋白动态)
Chemotaxis (趋化性)
Background
Directed migration along gradients of chemicals (chemotaxis) is fundamental to many developmental and physiological processes. Immune cells represent prototypical migratory cells that rely on active motion to search tissues, find pathogens, and interact with other cells in order to launch immune responses. Among these cells, neutrophils are the first to be recruited to injured or infected tissues from the bloodstream [1]. Their chemotaxis to these inflammatory sites involves changes in cytoskeletal dynamics in response to gradients of chemicals produced therein, including primary chemoattractants produced by microbes or damaged cells (e.g., formyl peptides) or secondary attractants (i.e., attractants produced by inflamed tissue cells or by neutrophils themselves, such as chemokines and leukotriene B4) [1,2]. Understanding neutrophil chemotaxis mechanisms is important for ultimately controlling the accumulation of these cells in tissues in inflammatory disease settings, but also for providing mechanistic paradigms for understanding how other types of cells undergo directional motion in general.
Neutrophil motility and gradient sensing have been studied in vitro in settings where gradients can be administered in a controlled fashion. For example, assays where micropipettes are introduced to cells during imaging are very useful for inferring causal effects of gradients on cell behaviour. In addition, a variety of microfluidic setups can be used to generate long-lived gradients on 1D, 2D, or 3D migration devices, where the movement of the cells can be monitored over time [3–6]. Relatively few assays have been developed to profile neutrophil chemotaxis and actin dynamics in response to gradients in vivo. For example, injection of chemoattractants is difficult to perform in vivo during imaging.
Here, we provide a protocol for implementing laser-assisted tissue injury to profile the behaviour and actin dynamics of neutrophils in response to gradients. Wounds introduce a cocktail of damage-induced chemoattractants, and the behaviour of cells can be followed in response to such endogenous tissue-derived gradients [7,8]. Whilst the profile of gradients is relatively complex and difficult to determine, the advantage of this approach is that cells can be profiled in an intact organism. To compare with unspecific motion dynamics before gradient exposure, wounds can be performed in an anatomical site where neutrophils are already present and motile (such as the cephalic mesenchyme region of the zebrafish larva), or cell motion can be pre-stimulated in the tissue of interest [9]. This enables the determination of changes in the same moving cell before and after gradient exposure.
The approach has certain advantages over other intravital methods. Two-photon laser ablation is advantageous over one-photon ablation, offering more precise wounding and less background photodamage, as only the focal plane is exposed to a high number of photons [10,11]. Furthermore, the use of near-infrared light allows the performance of wounding deeper into tissues [10]. The use of the transparent zebrafish larva, as opposed to a mouse, simplifies the experimental approach and enables higher-resolution imaging of sub-cellular scale dynamics. In terms of further applications, the focal nature of the laser wound makes the assay suitable for studies of neutrophil swarming, notably when performing these wounds near or on the caudal hematopoietic tissue, where neutrophil density is highest [12]. In addition, the assay can be adapted to visualise host–pathogen interactions during wound healing, by the inclusion of microbes in the medium [12]. Genetic or chemical inhibition experiments can be used in conjunction with this assay to investigate signalling mechanisms [9,12].
Materials and reagents
Biological materials
Transgenic zebrafish: Tg(mpx:Lifeact-Ruby)
Promoter: Lysozyme neutrophil-specific promoter
Specificity: Lifeact (17 amino acids of yeast Abp140) fused to Ruby, which detects all F-actin, as described in Riedl et al. [13].
Reagents
4.53% NaOCl (Cleanline, catalog number: CL3013)
Methylene blue (Sigma-Aldrich, catalog number: M9140-25G)
1-phenyl-2-thiourea (PTU) (Sigma-Aldrich, catalog number: P7629-25G)
3-amino benzoic acid ethyl ester (MS-222 or Tricaine) (Sigma-Aldrich, catalog number: E10521-50G)
Low-melting point (LMP) agarose (Invitrogen, catalog number: 16520)
Leukotriene B4 (LTB4) (Sigma-Aldrich, catalog number: L0517)
Sodium chloride (NaCl), certified AR for analysis (Fisher Chemical, catalog number: S/3160/60)
Potassium chloride (KCl) (Honeywell Riedel-de. Haën, catalog number: 31248)
Calcium chloride 2-hydrate (CaCl2·2H2O) (AnalaR, catalog number: 100703H)
Magnesium sulphate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M2773)
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), bioPerformance certified (Sigma-Aldrich, catalog number: H4034)
Trizma base (Sigma-Aldrich, catalog number: T6066)
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 06203)
Solutions
E3 embryo medium (see Recipes)
MS-222 solution (see Recipes)
PTU stock solution (see Recipes)
LMP agarose solution (see Recipes)
Recipes
E3 embryo medium solution
Reagent Final concentration (for 1×) Quantity (for 60×) NaCl 5 mM 17.2 g KCl 0.17 mM 0.76 g CaCl2.2H2O 0.33 mM 2.9 g MgSO4.7H2O 0.33 mM 4.9 g ddH2O n/a Up to 1 L Total n/a 1 L E3 can be made up as a 60× stock and stored at room temperature. To prepare 60×, weigh each salt as per the table above and include 10 g of HEPES per 1 L as buffering agent. Add ~800 mL of ddH2O, adjust the pH to 7.8, and then top up with ddH2O to a final volume of 1 L. To make a 1× solution, dilute 16.7 mL of 60× solution and 1 mL of 0.01% methylene blue into a final volume of 1 L of ddH2O.
MS-222 solution
Reagent Final concentration (for 1×) Quantity (for 25×) MS-222 0.16 mg/mL 400 mg ddH2O n/a Up to 100 mL Total n/a 100 mL MS-222 can be made up as a 25× stock and stored at -20 °C for long-term storage or at 4 °C for short-term storage. To make 25× stock, weigh the corresponding amount of MS-222 as per the table above, add ~80 mL of ddH2O, adjust the pH to 7.5 using 1 M Tris pH 9, and then top up with ddH2O to a final volume of 100 mL.
PTU solution
Reagent Final concentration (for 1×) Quantity (for 50×) PTU 0.003% (w/v) 1.5 g ddH2O n/a 1 L Total n/a 1 L PTU can be made up as a 50× stock and stored in the dark at room temperature. PTU is not very soluble at room temperature. Therefore, stirring and mild heating at 60 °C is recommended to dissolve.
LMP agarose solution
Reagent Final concentration Quantity LMP agarose 1.2% (w/v) 0.24 g 1× E3 n/a 20 mL Total n/a 20 mL Use fresh LMP agarose to ensure polymerisation.
Laboratory supplies
1.5 mL TubeOne microcentrifuge tubes (Starlab, catalog number: S1615-5500)
Aquafine shader sable watercolour brush size 4/0 (Daler-Rowney, catalog number: 282034040)
Nunc glass-bottom dishes (Thermo Scientific, catalog number: 150680)
Fine point, non-sterile disposable transfer (Pasteur) pipettes (Liquipette, catalog number: 127-P406-000)
3 mL non-sterile disposable transfer (Pasteur) pipettes (Liquipette, catalog number: 127-P503-000)
50 mL skirted, sterile conical tubes (CellStar, catalog number: 210261)
15 mL sterile conical tubes (CellStar, catalog number: 188271)
Equipment
Incubator LMS series 2 (Wolflabs, catalog number: 220)
Techne FDB02AD DB-2A Analogue Dri-Block® heater (Techne, catalog number: 36620-12)
Analytical balance (Sartorius, catalog number: TE412)
pH meter with InLab® Routine Pro-OSM pH electrode (Mettler Toledo, catalog number: 51344055)
Olympus MVX10 Macro zoom fluorescence upright microscope (Olympus Life Science, catalog number: MVX10) equipped with pE-300 white LED microscope illuminator (CoolLED, catalog number: pE-300 white) to screen embryos for transgenic expression
Zeiss Stemi 2000-C stereomicroscope to remove unfertilised eggs and unhealthy or dead embryos (Carl Zeiss, catalog number: Stemi 2000-C)
TriM Scope II Multiphoton microscope (LaVision BioTec TriM Scope II) for laser wounding and live-cell imaging
Software and datasets
ImSpector Pro software (5.0.284.0, LaVision Biotec, © 1998-2016) for image acquisition on TriM Scope II – Multiphoton microscope
Fiji (ImageJ 1.52p, June 2019, publicly available [14]) for generation of maximum intensity z-projection of the 3D image datasets and generation of individual timelapse video for each neutrophil we track
MATLAB (R2018b, Sept 2018, requires a license) for image analysis and data plotting
Procedure
文章信息
版权信息
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Williantarra, I., Georgantzoglou, A. and Sarris, M. (2024). Visualising Neutrophil Actin Dynamics in Zebrafish in Response to Laser Wounding Using Two-Photon Microscopy. Bio-protocol 14(11): e4997. DOI: 10.21769/BioProtoc.4997.
- Georgantzoglou, A., Poplimont, H., Walker, H. A., Lämmermann, T. and Sarris, M. (2022). A two-step search and run response to gradients shapes leukocyte navigation in vivo. J Cell Biol. 221(8): e202103207.
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分类
免疫学 > 免疫细胞成像 > 双光子显微镜技术
细胞生物学 > 细胞成像 > 共聚焦显微镜
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