We thank Christine Holt and Bill Harris for help with confocal microscopy. We thank Darren Gilmour for the fluorescent timer backbone constructs and Anna Huttenlocher for the Tol2-Lyz backbone vector. We thank Steve Renshaw for the Tg(mpx:GFP)i114 line. This work was supported by the MRC (MR/L019523/1), the Wellcome Trust [204845/Z/16/Z]; Isaac Newton Trust [12.21(a)i] and an Isaac Newton Trust grant 19.23(n). C.C. was supported by an MRC DTP studentship and partly by the Wellcome Trust [204845/Z/16/Z]; Isaac Newton Trust [12.21(a)i] grant. H.W. was supported by an MRC DTP Studentship. H.P. was supported by a Wellcome Trust PhD grant (105391/Z/14/Z) and partly by a Wellcome Trust [204845/Z/16/Z]; Isaac Newton Trust [12.21(a)i] grant and the MRC (MR/L019523/1).

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+zebrafish+line+for+in+vivo+visualisation+of+neutrophil+myeloperoxidase.">A transgenic zebrafish line for in vivo visualisation of neutrophil myeloperoxidase.</a> PLoS One. 14 (4), 0215592 (2019).</li><li>Coombs, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+receptor+trafficking+coordinates+neutrophil+clustering+and+dispersal+at+wounds+in+zebrafish.">Chemokine receptor trafficking coordinates neutrophil clustering and dispersal at wounds in zebrafish.</a> Nature Communications. 10 (1), 5166 (2019).</li><li>Hall, C., Flores, M. 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The authors declare no conflict of interest

The method described allows live imaging of receptor dynamics in response to endogenous ligands in situ during an inflammatory response to tissue damage. The use of Cxcr1/Cxcr2 neutrophil reporters could be expanded to other physiological settings, such as infection, tumor models or other types of tissue damage14,25,26,27. In addition, transgenic rescue lines, in which the endogenous receptor is suppressed and rescued by an exogenous mutant receptor, could provide useful tools to dissect the importance of specific neutrophil migration patterns in immune responses. For example, Cxcr1 receptor mutants that have impaired desensitization cause more prominent neutrophil clustering at inflammatory sites14. This gain of function phenotype could be used to understand the role of neutrophil congregation in different physiological processes, e.g., wound repair, infectious disease, or tumor evolution, and complement receptor knockdown/knockout experiments. The methodology also provides a basis to expand the range of available reporters. The choice of fluorescent reporter is important to consider and depends on the biological question. We found that the constitutive turnover of these chemokine receptors in neutrophils was high, in comparison to epithelial cells, and that reporters with fast maturation (e.g., sfGFP) were required to report membrane levels at steady state and resolve differences upon neutrophil stimulation8,14. Thus, membrane ratios of sfGFP/tagRFP are not applicable for measuring ligand-induced internalization in this cell type, but the pattern of tagRFP allows tracking of the intracellular fates of the receptor, which could be useful in some studies. We also found that the more concentrated intracellular signal of tagRFP is useful for screening individual larvae. An alternative approach for measuring receptor levels at the plasma membrane would be to co-express a fluorescent membrane marker in neutrophils either in the same transgene9 or in an independent transgene14. In the former scenario the transgene would provide an additional means for screening the fish and expression levels would be comparable between the marker and the receptor. The latter approach would be more modular, in that a zebrafish line with a receptor reporter could be combined with different reporter lines. In either case, it is worth noting that membrane quantification of the receptor levels is challenging in clustered neutrophils (see below). Finally, we note that a possible extension of this protocol would be to follow up the live imaging by immunohistochemistry for more detailed localization analyses.
The Tol2 transgenesis system is well established7 and the lysozyme C promoter has been used extensively for neutrophil expression11,15. The transgenesis approach is, therefore, relatively straightforward and the expression level achieved with this promoter is high enough to provide sufficient contrast for analysis of receptor dynamics. A possible limitation is that the expression level does not recapitulate endogenous receptor expression levels. New CRISPR technologies could be deployed to establish knock-in lines for receptors of particular interest28. These technologies are still cumbersome and may not guarantee the required expression levels for subcellular imaging, but their successful development would be an important breakthrough for understanding endogenous signaling dynamics. Functional validations are important for interpreting data with transgenic receptor constructs. For example, ligand recognition assays can be used to establish that the fluorescent fusion protein is functional and rescue of knockout phenotypes could be used to establish that the transgenic neutrophil expression levels are compatible with functionality14. Finally, a more direct way to validate the receptor fusion would be to utilize an in vitro functional assay with labelled receptor alongside non-labeled versions14.
The quantification approach addresses specific difficulties in accurate membrane segmentation in neutrophils in vivo. In cells of epithelial nature, quantification of receptor levels can be executed automatically by normalizing membrane receptor levels to a control marker, which can be expressed in tandem or separately9. Indeed, we have applied such an approach, when using the ligand-recognition assay in gastrulating embryos14. However, neutrophils undergo complex, rapid changes in cell shape in vivo, making the membrane segmentation difficult both in 2D and 3D14. This is even more challenging when neutrophils cluster, which occurs in many physiological settings29. The contrast metric overcomes this limitation as it does not require membrane segmentation but instead reflects the overall state of receptor distribution in the cell (membranous/smooth vs vesicular/dotty). It is important to note that contrast metric can be affected by the overall contrast of the image, so normalization of individual cell values to an internal reference is required to account for variability of signal in different embryos/samples. For example, we used the mean cell contrast value of non-responsive neutrophils in the CHT (i.e., neutrophils that remain stationary and do not migrate into the ventral fin)14. An additional possibility would be to normalize with contrast values of a control marker in the same cell. This would provide a solution when an internal reference of non-responding cells is not available and may likely resolve finer quantitative differences in receptor dynamics between different conditions.
The location of imaging is another variable to consider. The reason for choosing the ventral fin wound here, as opposed to the more commonly used tail fin wound model16,30, is because the site of wounding is nearby the site of neutrophil residence/migratory origin. This accelerates the timeline of the assay, as it takes relatively little time for neutrophils to arrive. Additionally, it provides the opportunity to capture cell behavior both at the migration origin (CHT) and the target location of interest (wound). This is relevant here, because the spatial and temporal resolution required for subcellular imaging is difficult to combine with a large field of view or multi-position scanning. Thus, the ventral fin wound assay permits tracking of the evolution of the migratory response from the migration origin and simultaneous capturing of unspecific receptor fluctuations in cells that do not respond. As mentioned above, the latter is useful for quantification purposes as it provides an internal reference for unspecific dynamics. In other systems, it may not be possible to have such an internal reference, in which case the contrast values of a co-expressed membrane marker would provide an alternative control.
In summary, we anticipate that the methodology is applicable to other systems and can be deployed for a variety of purposes. For example, the same reporters could be utilized in other inflammatory settings, such as infection settings or other disease models. The repertoire of zebrafish receptor reporter lines could be expanded to other signaling receptors, to understand signaling mechanisms or report ligand dynamics in vivo. The approach can be combined with knockdown/knockout techniques to interrogate the mechanistic basis of observed dynamics. For example, perturbation of ligand expression can indicate the ligand dependency for observed receptor dynamics. In the future, we envisage that the system could be further refined to incorporate knock-in insertion of reporters. Ultimately, findings using this methodology would provide novel insights valuable beyond the zebrafish community, given the conservation of these signaling receptors in mammals and the relative challenge of conducting these studies in larger organisms.

Leukocyte migration is of paramount importance for immune responses. Immune cells are prototypical migratory cells, which are remarkably capable of traversing tissues and blood vessels and sensing a range of chemical guidance cues to migrate directionally towards microbes or other host cells of importance. Correct guidance relies on the recognition of chemoattractants, among which chemokines represent the most prominent category1. Chemokines are recognized by highly specific seven-transmembrane G protein coupled receptors. Upon chemokine binding, chemokine receptors change conformation leading to the activation of associated trimeric G proteins and their dissociation into functional signaling subunits that promote cytoskeletal changes and directed migration1. Secondarily, chemokine receptors are phosphorylated, and this modification leads to desensitization to attractant, which can be followed by rapid re-sensitization/recycling or intracellular degradation and down-regulation from the cell surface2. These receptor dynamics influence the duration and dose of signaling received by the cells but how they affect leukocyte migration behavior has been difficult to elucidate in vivo.
Tracking receptor dynamics in live leukocytes in traditional mammalian systems faces several challenges. For live studies, receptor fusions with fluorescent proteins must be expressed in the cells. This is challenging in primary leukocytes, particularly in neutrophils, and studies so far have used surrogate neutrophil cell lines to express chemokine receptors3,4. Generation of transgenic mouse models, in which leukocytes express a fluorescent receptor or mutant receptors with informative trafficking defects5,6, entails considerable investment of time and resources. Even in these instances, the imaging resolution and contrast for imaging receptor dynamics in the live animal can be limited and studies have used immunohistochemistry on fixed tissue sections5. Given these technical challenges, our understanding of how chemoattractant receptors dynamics affect cell behavior in a live tissue setting is currently limited.
Here we provide a methodology to monitor receptor trafficking in zebrafish neutrophils. Zebrafish are genetically tractable, like mice, but transgenesis is relatively more straightforward through the use of efficient transposon systems and direct zygote manipulation7. The transparent larva is ideally amenable to imaging. The chemokine receptor dynamics have been visualized in primordial germ cells and the lateral line primordium by expression of corresponding fusions with fluorescent reporters8,9,10. Zebrafish larvae are equipped with mature neutrophils that have highly conserved genetic and cellular properties with respect to mammalian neutrophils. Subcellular signaling dynamics such as cytoskeletal dynamics and polarity regulators have been visualized in these cells by the generation of corresponding transgenic lines11,12,13. Recently, we visualized and functionally analyzed chemokine receptor dynamics in neutrophils during the course of inflammatory responses to tissue damage14. Here, we describe the generation of transgenic reporter lines for chemokine signaling in neutrophils, preparation of embryos for live imaging, a wound assay for studying neutrophil signaling and the protocol for acquisition and analysis of images. We also provide a side-protocol to test chemokine receptor responses to candidate ligands, which is useful when trying to establish ligand recognition patterns in uncharacterized receptors. These techniques can be used in combination with further genetic manipulations, such as inhibition of endogenous chemokine expression or generation of mutant receptors with altered ligand-induced trafficking, to interrogate how specific signaling dynamics affect leukocyte behavior in vivo. The transgenic lines expressing fluorescently tagged chemokine receptors can also be used as reporters for endogenous chemokine gradients, which are otherwise difficult to detect by direct antibody staining. The described methodology provides scope for expanding the generation of reporters to other immuno-signaling receptors.

<table>
	<tbody>
		<tr>
			<td>1-phenyl-2-thiourea</td>
			<td>Sigma-Aldrich</td>
			<td>P7629-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL CellStar Centrifuge Tube</td>
			<td>Grenier Bio-One Limited</td>
			<td>210270</td>
			<td></td>
		</tr>
		<tr>
			<td>Clark capillary glass</td>
			<td>Harvard Apparatus</td>
			<td>30-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleanline Thin Bleach</td>
			<td>Scientific Laboratory Supplies</td>
			<td>CLE0301</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scope</td>
			<td>Nikon</td>
			<td>SMZ645</td>
			<td></td>
		</tr>
		<tr>
			<td>Dri Block heater</td>
			<td>Techne</td>
			<td>FDB02AD</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>National Institutes of Health, Bethesda, MD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, Jewelers Dumont No. 5</td>
			<td>Sigma-Aldrich</td>
			<td>F6521</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom microwell dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pasteur pipette</td>
			<td>Fisherbrand</td>
			<td>11795098</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Ambion mMESSAGE mMACHINE SP6 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>10391175</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 confocal microscope</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melting point agarose</td>
			<td>Invitrogen</td>
			<td>16520-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Narishige</td>
			<td>GJ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>The MathWorks, Inc., Natick, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M-2643</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection system</td>
			<td>Parker</td>
			<td>Picospritzer III</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader pipette tips</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>M-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>Flaming/Brown P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Thermo-Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>PerkinElmer Spinning Disk UltraVIEW ERS, Olympus IX81 spinnng disk confocal microscope</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (120mm)</td>
			<td>Grenier Bio-One Limited</td>
			<td>632180</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>P0290-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(A) Tailing Kit</td>
			<td>Invitrogen</td>
			<td>AM1350</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small paintbrush</td>
			<td>N/A</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectral Applied Research LMM5 laser module</td>
			<td></td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scalpel Blade No. 23</td>
			<td>Swann-Morton</td>
			<td>233-5528</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine MS222</td>
			<td>Sigma-Aldrich</td>
			<td>E10521-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Volocity software</td>
			<td></td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Yokogawa CSU10 spinning disc confocal scanner</td>
			<td>Yokogawa</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of chemokine receptors in zebrafish neutrophils during wound responses

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

Ventral fin wounding is followed by rapid neutrophil mobilization from the CHT into the ventral fin and clustering at the wound margin, within 30-60 min (Figure 1). We visualized the distribution of two chemokine receptors, Cxcr1 and Cxcr2, which are expressed by zebrafish neutrophils24 and recognize Cxcl8a and Cxcl8b14, using spinning-disk confocal microscopy. We generated two corresponding transgenic lines, Tg(lyz:Cxcr1-FT) and Tg(lyz:Cxcr2-FT), in which neutrophils express a fluorescent timer (FT) construct of the receptor, i.e. a fusion with a tandem of sfGFP and tagRFP (Figure 2 and reference14). The use of the two fluorophores was intended to allow monitoring of a broad range of receptor fates and provide estimates of protein turnover time at the plasma membrane, as newly synthesized receptors would fluoresce in green and progressively become red as they age8,14. However, these receptors were found to have fast constitutive turnover at the neutrophil plasma membrane and that the residence time was shorter than the maturation time of tagRFP, with sfGFP showing membrane localisation and tagRFP showing vesicular localization at steady state (Supplementary Video 1 and ref14). Therefore, we focused on the distribution of sfGFP to monitor ligand-induced internalization at sites of tissue damage. The pattern of receptor distribution was quantified using the contrast metric, which reports differences in intensity between neighboring pixels. The rationale is that when the receptor distribution is membranous and smooth, the contrast value is low. When the receptor distribution is vesicular and more punctate, then the contrast value is high (Figure 3).
An alternative method is to quantify the ratio of receptor levels (sfGFP intensity) over the levels of a control membrane marker e.g. membrane CFP (mCFP) (Figure 3). Both methods could detect receptor internalization, as indicated by more vesicular receptor distribution pattern globally in the cell (higher contrast value) or lower receptor levels at the membrane (lower sfGFP/mCFP ratio). However, the contrast metric could also detect receptor internalization in neutrophil clusters at the wound, in which membrane segmentation was less accurate and not applicable (Figure 3). Using this metric, we were able to quantify visible differences between Cxcr1 and Cxcr2 trafficking in neutrophils at wounds (Figure 4 and Supplementary Video 2). Cxcr1-FT internalized in cells located at the wound whereas Cxcr2-FT remained membranous in neutrophils at the wound (Figure 4A-C, Supplementary Video 2 and Supplementary Video 3). Suppression of Cxcl8a and Cxcl8b, through morpholino treatment, differentially affected Cxcr1-FT internalization at wounds (Figure 4C,D). To further validate that Cxcr1-FT responds to Cxcl8a, we performed chemokine response assays in early embryos. We found that Cxcr1-FT markedly internalized in embryos in which Cxcl8a was co-expressed (Figure 5). Altogether these results indicate that the described methods can be deployed to measure chemokine-induced receptor internalization in neutrophils and establish the identity of the ligand mediating these effects.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61679/61679fig01.jpg" /> 
Figure 1: Neutrophil migration to ventral fin wounds.&#160;(A) (Left) Cartoon of 3 dpf larva showing the location of the caudal hematopoietic tissue (CHT), the venus circulation (VC, blue), the ventral fin (VF) and the wound site. (Right) Cartoon depicting the area of the wound (W) with neutrophils getting mobilized from the CHT and clustering at the wound. The caudal vein plexus (CVP) of the CHT tissue is drawn in blue. (B) Bright field image (left) and confocal projection (right) showing the ventral fin wound and the distribution of neutrophils in Tg(mpx:GFP) larvae at 2 h post-wounding. Dashed lines show VF and CHT outlines. Scale bar = 25 &#181;m. Cartoon and fluorescent image modified from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://www.jove.com/files/ftp_upload/61679/61679fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61679/61679fig02.jpg" /> 
Figure 2: Live imaging of chemokine receptor trafficking in neutrophils.&#160;(A) Constructs used for neutrophil-specific transgenic expression of Cxcr1-FT (Fluorescent Timer) and Cxcr2-FT. Confocal projections of neutrophils in the head of a 3 dpf transgenic larva (Tg(lyz:Cxcr1-FT), top; Tg(lyz:Cxcr2-FT), bottom) showing tRFP (magenta) and sfGFP (green) channels. Scale bar = 20 &#181;m. (B) Anatomical scheme of 3 dpf larva as in Figure 1A. Below the larva are schemes depicting the area of the wound (W) with neutrophils getting mobilized from the CHT (top) or performing chemotaxis upon entering the ventral fin (bottom). Dashed square indicates area imaged in snapshots on the right. (C) Neutrophils in Tg(lyz:Cxcr1-FT) larvae (sfGFP is shown) upon mobilization from the CHT (top panels) or chemotaxis towards the wound (bottom panels). Arrows show the same cells over time. Time points on the right image are minutes elapsed after image on the left. Scale bar = 10 &#181;m. (D) Schematic representation of experimental approach for live imaging of chemokine receptor trafficking. Panels A-C modified from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://www.jove.com/files/ftp_upload/61679/61679fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61679/61679fig03.jpg" /> 
Figure 3: Quantification examples of receptor dynamics.&#160;Single (blue) or clustered neutrophils (green) at wounds or non-mobilized neutrophils in the CHT (red, orange) were segmented and analyzed by different methods to compare results. The same example cells shown were analyzed with two methods to relate what is seen in the image with the range of values extracted. (A) The surface of the selected, example cells were segmented based on contour definition in the sfGFP channel. (B) Contrast was computed from the example cells shown in A. (C) The membrane of the selected, example cells were segmented based on contour definition in the CFP channel. Ratiometric analysis of sfGFP/CFP followed. (D) The ratio of sfGFP/CFP was computed on the example cells shown in C. Error bars represent S.E.M. from individual cells, in cases of n&#62;1, values here were not used for statistical analysis but merely to exemplify measurements obtained with the different quantification methods. Scale bar = 10 &#181;m. Figure modified from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://www.jove.com/files/ftp_upload/61679/61679fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61679/61679fig04v2.jpg" /> 
Figure 4: Differential dynamics of Cxcr1 and Cxcr2 in response to wounding.&#160;(A) Confocal projection of neutrophils in Tg(lyz:Cxcr1-FT) or Tg(lyz:Cxcr2-FT) larvae at the wound at 80 min post-wounding (sfGFP channel shown). Scale bar = 10 &#181;m.&#160;&#160;(B) Magnified Cxcr1-FT neutrophil (left) and Cxcr2-FT (right) at the wound. Green receptor is shown in gray. Scale bar = 5 &#181;m. (C) Normalized contrast (contrast per individual neutrophil normalized to the mean contrast of non-mobilized cells in the CHT). cxcl8a refers to injection of a splice-blocking together with a translation-blocking morpholino for cxcl8a. cxcl8b refers to injection with a splice-blocking morpholino for cxcl8b. For Tg(lyz:Cxcr1-FT): n=24 cells (CHT), n=47 cells (wound) from 8 larvae. For Tg(lyz:Cxcr1-FT) with morpholinos: n=28 cells (Cxcl8a-MO) from 5 larvae, n=16 cells (Cxcl8b-MO) from 5 larvae. For Tg(lyz:Cxcr2-FT): n=10 cells (CHT) and n=20 cells (wound) from 3 larvae. Data were pooled from independent larvae acquired in 1-5 imaging sessions. Kruskal-Wallis test with Dunn&#8217;s multiple comparisons test for Tg(lyz:Cxcr1-FT), two-tailed unpaired Mann-Whitney test for Tg(lyz:Cxcr2-FT). (D) Confocal projection of neutrophils in Tg(lyz:Cxcr1-FT) transgenic larvae treated with cxcl8a morpholino (MO) (left) and cxcl8b MO (right) responding to fin wounds (sfGFP channel shown in green). Snapshot taken at timepoints of equivalent neutrophil accumulation (85 min post-wounding&#160;in left image and 45 min post-wounding&#160;in right image). Scale bar = 10 &#181;m. Figure modified from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://www.jove.com/files/ftp_upload/61679/61679fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61679/61679fig05.jpg" /> 
Figure 5: Chemokine response assay in early embryos.&#160;Laser-scanning confocal slices of gastrulating embryos showing expression and distribution of Cxcr1-FT. 100 pg of Cxcr1-FT mRNA was injected into one cell-stage eggs with or without 150 pg Cxcl8a mRNA. Green and red receptors are shown in separate channels. Control membrane CFP marker (mCFP) is shown in the cyan channel. Scale bar = 20 &#956;m. Figure modified from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://www.jove.com/files/ftp_upload/61679/61679fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Movie 1: Transgenic neutrophils in the head of a Tg(lyz:Cxcr1-FT) (left) and Tg(lyz:Cxcr2-FT) (right) larva at 3 dpf. sfGFP(green), tagRFP (magenta). Frame interval is 30 sec and frame rate is 5 fps. Scale bar = 20 &#956;m. Video originates from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary_Movie1.avi" target="_blank">Please click here to download this video.</a>
Supplementary Movie 2: Neutrophils in Tg(lyz:Cxcr1-FT) (left) and Tg(lyz:Cxcr2-FT) (right) transgenic larvae responding to fin wounds. Movie starts within 10 min post-wounding&#160;and lasts 60 min. sfGFP (green), tagRFP (magenta). Frame interval is 30 sec and frame rate is 10 fps. CHT = caudal hematopoietic tissue. VF = ventral fin. Scale bar = 25 &#956;m. Video originates from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary_Movie2.avi" target="_blank">Please click here to download this video.</a>
Supplementary Movie 3. Additional examples of neutrophils from a wounded Tg(lyz:Cxcr1- FT) transgenic larva (different larva to that shown in Video 2), acquired at higher resolution, showing receptor internalization (sfGFP channel shown in green) upon mobilization in the CHT or upon entry and chemotaxis in the ventral fin. Frame interval is 30 sec and frame rate is 2 fps. Scale bar = 10 &#956;m. Video originates from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary_Movie3.avi" target="_blank">Please click here to download this video.</a>
Supplementary File 1: <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary file 1.m" target="_blank">Please click here to download this file.</a>
Supplementary File 2: <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary file 2.m" target="_blank">Please click here to download this file.</a>
Supplementary File 3: <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary file 3.m" target="_blank">Please click here to download this file.</a>
Supplementary File 4: <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary file 4.m" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses at JoVE.com

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

Here we describe protocols to perform live imaging and quantitative analysis of chemoattractant receptor dynamics in zebrafish neutrophils

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...

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Research

JoVE Journal

Immunology and Infection

2.4K Views.  University of Cambridge. Here we describe protocols to perform live imaging and quantitative analysis of chemoattractant receptor dynamics in zebrafish neutrophils

Video: Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.