Name: Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy (Video) | JoVE Journal
Uploaded: 2011-06-02T12:00:00.000+00:00
Duration: 8 min 50 s
Description: Learn to visualize neutrophil phagocytosis and NET formation in infected lungs using 2-photon microscopy. This technique enables real-time observation of immune cell dynamics in live tissue.

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5 minutes

The authors would like to thank Dr. Lars Philipsen for help with optimizing the intravital movies, Dr. Jonathan Lindquist for carefully reading the manuscript, and all members of the Gunzer laboratory for helpful discussions and comments during the development of the method. This work was funded by grants from the Deutsche Forschungsgemeinschaft (DFG, SFB 854) to M.G.

1. Infection
<ol>
	<li>Swollen conidia of the mold Aspergillus fumigatus (strain ATCC 46645) are generated by pre-incubation in RPMI 1640 (Biochrom AG, Berlin, Germany) 
	supplemented with 5% FCS (v/v). 2x107 resting conidia are resuspended in 5 ml medium and incubated in a 6-well tissue culture plate (TPP AG; Trasadingen, 
	Switzerland, catalog # 92006) for 7h at 37°C.</li>
	<li>Following the swelling period, the spores are fluorescently labeled by adding 10 μl of calcofluor white stock solution (Sigma Aldrich, Deisenhofen, Germany, 
	catalog # F3543; [25 mg/ml] in DMSO) to each Aspergillus containing well in the 6-well plate, reaching a final calcofluor concentration of 50 μg/ml RPMI. 
	Following gentle mixing, the spores are incubated for an additional 15 min at 37 °C. </li>
	<li>In the next step, the spores are harvested by filtration through a 70 μm cell strainer (BD Biosciences, Heidelberg, Germany). After washing once 
	with 10 ml PBS (centrifuge at 900xg for 5 min at room temperature) the pellet is resuspended in 2 ml PBS and the total number of swollen conidia 
	determined by counting with a Neubauer chamber. Finally the spore suspension is spun down at 900xg for 5 min at room temperature, the 
	supernatant is carefully removed, and the pellet resuspended to the final concentration of 1x107 spores per 100 μl PBS.</li>
	<li>For infection, 100 μl of spore solution is applied intratracheally into female mice (C57/BL6, 8–10 weeks old, Harlan, Germany). If neutrophils are to be 
	observed as well, the use of Lys-EGFP mice, carrying EGFP under the lysozyme promotor1 is recommended. Begin by anesthetizing the animal with a single i.p. 
	injection of 150 μl Ketamin/Rompun solution (Inresa Arzneimittel GmbH, Freiburg, Germany (Ketamin) and Bayer Vital GmbH, Leverkusen, Germany (Rompun), 1 ml 
	Ketamin [50 mg/ml] + 0.5 ml Rompun [2 %] + 3.5 ml sterile NaCl [0.9 %]). 5 minutes later the anesthetized mouse can be fixed with an elastic band by its teeth 
	to the ramp (i.e. a sloped surface) to facilitate the intubation (supplemental figure 1). Using forceps, the tongue is pulled to one side and a 22G indwelling 
	venous catheter (B. Braun AG, Melsungen, Germany, Vasofix Braunüle) can be gently inserted into the trachea under permanent illumination with a goose neck 
	lamp. The successful insertion of the catheter is verified by observing regular movement of the animal's thorax with the frequency of the mechanical 
	ventilator. When the successful intubation has been confirmed, 100 μl spore suspension is applied using a 100 μl micropipette. This volume should be 
	inhaled by the animal without any additional help in 1-2 s. An enhanced distribution of fungal particles inside the lung is achieved by mechanically 
	ventilating the infected animal for 2 minutes with a small animal respirator (MiniVent, Hugo Sachs, March-Hugstetten, Germany) at rate of 250 breaths 
	per minute and an inhalation volume of 300 μl per breath (supplemental figure 2). After the infection animals are returned to their cage and observed every 5 minutes until ambulatory again. Mice do not show any evidence of pain or distress during the following incubation period.</li>
</ol>
2. Lung preparation
<ol>
	<li>7 h after infection with the spores, the animal is euthanized by an overdose of isoflurane (Baxter GmbH, Unterschleiβheim, Germany) until movements and 
	breathing have ceased for more than 30 seconds. This is then followed by thoracotomy as a secondary method to assure clinical death and allow for visualization 
	and access of the trachea and lungs. Then the chest is carefully opened to expose the lung and the upper respiratory tract. Then another 22G indwelling venous 
	catheter is inserted into the trachea starting from the exposed epiglottis. Through this tubule the lungs are filled with 1 ml pre-warmed low melting agarose 
	(2 % w/v, Promega, Mannheim, Germany) using a 1 ml Omnifix syringe (B.Braun). As soon as the lungs are filled the trachea is tied off with a short piece of 
	sewing thread and the whole animal is put into a refrigerator at 4 °C. </li>
	<li>15 min later, when the agarose has solidified, the whole lung is excised beginning from the trachea. Using a pair of sharp pointed scissors, 
	the right lung lobe is subsequently removed, the bottom surface is briefly dried on a sheet of tissue paper and then the whole lobe is glued to the 
	preparation block of a vibratome (Campden Instruments, UK, 752M Vibroslice) using a drop of tissue adhesive for fixation (Carl Roth GmbH, Karlsruhe, 
	Germany, Roti-Coll 1). After 1 min the block is installed in the cutting chamber of the vibratome, which is filled with pre-chilled (4°C) PBS. The 
	vibratome is set to a mid-range vibration (5 out of 10 max.) and also moderate speed (5 out of 10 max.) such that a horizontal cross section of the lung 
	is produced. The upper half of the organ is then transferred into a small plastic Petri dish (5 cm diameter) where it is inverted and fixed with a self-made 
	flat washer (1 cm inner diameter) that is covered by a set of parallel nylon threads, each 1 mm apart (supplemental figure 3). Finally the Petri dish is 
	filled with 10 ml PBS supplemented with 10 μl of the DNA dye Sytox Orange [5 mM] (Invitrogen, Germany) resulting in a final dye concentration of 5 μM.</li>
</ol>
3. 2-photon laser microscopy
<ol>
	<li>The Petri dish containing the lung sample is then installed under the microscope objective so that the buffer can be warmed to 37°C by a temperature 
	sensor-controlled heater (supplemental figure 4). 2-photon microscopy is then carried out over the whole cut surface using a Zeiss LSM 710 NLO microscope 
	on an upright Axio Examiner stage equipped with a 20xNA1.0 water dipping lens (Zeiss, Jena, Germany). For imaging, different areas along the section are 
	scanned down to 400 μm depth using an illumination wavelength of 800 nm detecting green (530 nm) and red (580 nm) fluorescence, as well as the second 
	harmonic generation (SHG)-signal and the blue calcofluor fluorescence (at 400–470 nm emission) with external non descanned detectors (NDD). Besides 
	2-D images and time lapse movies (1 image every 5-10 seconds), single or repetitive 3-dimensional picture stacks can be subsequently rendered as 
	voxel renderings or single extended focus images or 4-D data (3D over time) using different software packages. This microscopic setup allows the 
	observation of cellular behavior in an almost intact environment that, as it constitutes the entry port for a variety of airborne pathogens, is 
	of enormous immunological relevance. Cell motility, phagocytosis and NET production by endogenous neutrophils following infection with the mold 
	Aspergillus fumigatus can be easily investigated under these in situ conditions.</li>
</ol>
4. Representative Results
If done properly the imaging will generate 2- or 3-color slides or movies. Although coloring is done in a post processing procedure and is thus freely adaptable, we generally select a coloring scheme that reflects the natural color of the dye/signal, which is detected in the relative channel. Thus, the Sytox dyes are depicted in red, the fungus as well as the SHG signal is shown in blue and, if present, EGFP-labeled cells are stained in green. In the first example (Fig. 1) the blue SHG signal depicts the tissue fibers of a non-infected lung and the red Sytox signal is produced by nuclei of lung-resident cells cut open by the vibratome. In the second example (Fig. 2) an infected lung is shown, where both, alveolar structures as well as fungal masses, appear in blue, while nuclei and NETs are stained red. The third example (Fig. 3) is from a Lys-EGFP transgenic animal where in addition to the blue and red structures the green neutrophils can also be seen. The migration of neutrophils and their phagocytosis of individual fungal elements in time lapse sequences is shown in the supplemental movie.
<img src="/files/ftp_upload/2659/2659fig1.jpg" alt="Fluorescence microscopy analysis: SHG, Sytox staining; cellular structure; non-infected specimen." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. The appearance of a non-infected lung slice in 2-color 2-photon microscopy. A lung slice was prepared from a non-infected C57/BL6 mouse and imaged as described in the protocol. Presented here are the SHG signal of the alveolar tissue structure (A), the Sytox signal of cell nuclei cut open during the preparation of the lung slice (B), and an overlay of the two channels (C). The boxed area in (C) is seen enlarged in (D). Please note the fibrous tissue at the bottom of the lung slice as compared to the clearly alveolar organization within the breathing-active areas of the lung above.
<img src="/files/ftp_upload/2659/2659fig2.jpg" alt="SHG/fungal imaging, sytox staining, overlay; microscopy, infected animal tissue study." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. The appearance of a lung slice of an Aspergillus infected wild type animal in 2-color 2-photon microscopy. A lung slice from a C57/BL6 mouse infected 7 h before with A. fumigatus was prepared and imaged as described in the protocol. Shown is the combined fungal structure and SHG of the alveolar tissue (A), the Sytox signal of DNA NETs as well as cell nuclei cut open during the preparation of the lung slice (B), and an overlay of the two channels (C). The boxed area in (C) is seen enlarged in (D). Please note, the clearly distinct areas of fungal masses, alveoli, and NET-structures are marked with the letters F, A and N, respectively.
<img src="/files/ftp_upload/2659/2659fig3.jpg" alt="Second harmonic generation microscopy image showing fungus, Sytox staining, and neutrophils interaction." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. The appearance of a lung slice of an Aspergillus infected Lys-EGFP animal in 3-color 2-photon microscopy. A lung slice of a Lys-EGFP mouse 
infected 7 h before with A. fumigatus was prepared and imaged as described in the protocol. Shown is the combined fungal structure and SHG of the alveolar tissue (blue), the Sytox signal of cell nuclei and NETs (red), as well as well as numerous neutrophils (green).
<img src="/files/ftp_upload/2659/2659supfig1.jpg" alt="rodent grip strength, strength measurement, experimental setup, lab research, physical endurance" data-altupdatedat="1788235205000" data-alt="Figure 4"> 
Supplemental figure 1. Mouse fixation for intubation. Under Ketamin/Rompun anesthesia the animal is fixed with an elastic band at its teeth in order to 
facilitate the intubation with a 22G indwelling venous catheter.
<img src="/files/ftp_upload/2659/2659supfig2.jpg" alt="Animal experiment setup with anesthetic equipment for behavioral study." data-altupdatedat="1788235205000" data-alt="Figure 5"> 
Supplemental figure 2. Mechanical mouse ventilation. The intubated mouse is infected by an i.t. application of 1x107 spores resuspended in 
100 μl PBS. An enhanced distribution of fungal particles inside the lung is achieved by mechanically ventilating the infected mouse with a small animal respirator.
<img src="/files/ftp_upload/2659/2659supfig3.jpg" alt="Metal test coupon for static analysis in material strength experiment, showing uniform strain lines." data-altupdatedat="1788235205000" data-alt="Figure 6"> 
Supplemental figure 3. Lung lobe fixation. After preparation of the right lung lobe the organ is fixed in a Petri dish by use of a laboratory-made 
flat washer that is covered by a set of parallel nylon threads.
<img src="/files/ftp_upload/2659/2659supfig4.jpg" alt="Optical microscopy setup; petri dish analysis; spectroscopy equipment; scientific observation." data-altupdatedat="1788235205000" data-alt="Figure 6"> 
Supplemental figure 4. 2-photon imaging setup. The Petri dish containing the right lung lobe is installed on a heating mat under the 2-photon microscope after addition of the DNA dye Sytox Orange.
Supplemental movie: Neutrophils migrating and phagocytosing fungal elements in Aspergillus infected lungs as seen by time lapse 2-photon microscopy of a 
living lung slice 7 h after infection with the fungus. Neutrophils are green, fungal elements and SHG are blue, and cell nuclei as well as NET-structures are depicted in red. The real time of the experiment is shown on the lower right. The scale bar depicts 50 μm.<a href="https://www.jove.com/files/ftp_upload/2659/5_9_1%20-%20supplemental.mpg">Click here to watch video</a>

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We have nothing to disclose.

Real-time 2-photon microscopy in vivo or in intact organs has gained profound importance in studies dealing with the physiology of immune cells over the past 10 years. It was with this technique that important events like the dynamics of T-cell activation within the lymph nodes first became visible2-4. More recently, researchers have also started to analyze specific cellular functions like the first steps in the generation of effector cells in lymphatic tissues using this approach5.
However, although a number of new biological concepts have been revealed using this method, there are still challenging and important 
questions for which no intravital visualization studies have been published thus far. Notably this applies to the mammalian lung. The interesting aspect of 
this organ, as entry port for a variety of airborne pathogens, makes it one of the most crucial surfaces at which immunological processes take place in the 
mammalian body. With every breath over the entire life span, unwanted particles are inhaled some of which have the potential to induce life threatening 
infections6. It is self-explanatory that at such a sensitive and endangered site a tight network of defense mechanisms needs to be present exhibiting 
the whole repertoire of immune responses. On the other hand it is very important that the induced immunological fight against potential pathogens at 
such a "dirty" place is tightly controlled. Exaggerated reactions of the immune system bear a high risk of harming the own body by massively injuring 
organ tissue upon stimulation of unspecific immune cell actions7,8.
In the light of these thoughts it would be extremely interesting and helpful to have the possibility to investigate cell behavior in 
mammalian lungs under true in vivo conditions. However, the fact that such a system has so far not been successfully implemented clearly points to the enormous 
difficulties that have to be solved in setting up a working protocol. The most demanding challenge probably is focus stability. The lung as the organ responsible 
for breathing is under constant movement in all three directions of space to realize inhalation. This circumstance alone causes severe imaging problems and can be 
considered an intravital imagers "nightmare". Already a slight motion to any dimension in space has a massive deteriorating effect on the microscopic view, which 
needs to be stable with micrometer precision in order to generate meaningful images9. Given its inherently tight local focus10, 2-photon microscopy is 
even more sensitive to focal instabilities, as a dislocation of a certain structure in the range of just a few micrometers in the Z-direction is 
equivalent to a complete loss of focus and thus a failed experiment.
The protocol presented in this study for the observation of immune cells within the murine lung is still not an in vivo 
application, but rather a close approximation to the situation in a functionally intact lung11. Ex vivo approaches for imaging lymphocytes, e.g. in 
explanted lymph nodes, have been shown to yield results equivalent to true in vivo observations12 and thus are highly relevant5. The in situ 
observations in lung slices, which are only possible with our approach, take place in an infected lung shortly after excision. The 3D integrity during the cutting 
process is ensured by the agarose matrix, an essential step to allow a precisely controlled cutting process of the lung. Although it is necessary to 
cool the explanted lung for a short period to allow a solidification of the agarose matrix, it is possible to return to near physiological conditions 
for cells after cutting and rewarming of the tissue. This is clearly shown by our data, which demonstrate that under these conditions neutrophils are 
highly active and exhibit their full potential as agile phagocytes, which are necessary for the effective clearance of an Aspergillus fumigatus 
infection. They patrol the lung tissue passing through epithelial barriers in order to reach the inner parts of alveoli and furthermore they 
actively take up fungal spores 11. A key finding of this work was the appearance of structures resembling Neutrophil extracellular traps (NETs) in 
the Aspergillus infected organ. NETs are a very recent finding of a novel defense mechanism in neutrophils13. However, since its initial description 
in 2004, the number of physiological or pathological conditions in animal models or humans where this phenomenon has been observed or is lacking has
been explosively increasing14-16. Interestingly, although so much work has been spent on these structures by so many different groups, most reports 
are still on a very descriptive level and not much is known about the mechanism of NET release and its regulation. With our protocol we were able 
for the first time to show NET fibers in an infected lung. Furthermore, we could demonstrate the importance of freshly recruited neutrophils as well 
as molecular fungal structures for their occurrence or inhibition11. This clearly shows the potential of our method to investigate the single 
steps of NET formation in more detail. One could think for example to use neutrophils from suitable knock-out mice to observe their ability of 
NET formation in adoptive transfer experiments.
Thus, although the direct observation of neutrophil immigration from the peripheral blood is not possible with this system due to the lack of 
blood supply after organ explantation, we still believe that our protocol is a valuable and relatively easy to handle approach that allows the imaging of early or 
late steps in the immune defense against lung infections. This is, therefore, an important step towards investigating this phenomenon within the breathing lung of 
live animals.

direct observation of phagocytosis and net-formation by neutrophils in infected lungs using 2-photon microscopy

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

The overall goal of this procedure is to visualize living neutrophil granulocytes and extracellular DNA nets in a lung that has been recently infected with a fungal pathogen. First mice that are positive for EGFP in their neutrophil population are infected with candia that are also fluorescently labeled. Next, the infection is allowed to develop over seven hours after which the infected animal is sacrificed.Then the lungs of the infected animal are filled with low melting agros to facilitate sectioning by beome. Finally, a thick slice of the infected lung tissue is acquired via viome and lung neutrophils. DNA nets and fungal elements are imaged.Ultimately, high resolution still images or movie sequences of granulocytes in live lung tissue can be obtained through multicolor two photon microscopy. The main advantage of this method over existing technologies is that the immune cells responsible for clearing the infection can be observed directly within the natural environment. This can be done during the process of net formation and fungal killing, and thus allows a much less artifactual analysis of neutrophil physiology.I invented this technique when Mathias annoyed me with his burning questions regarding the in vivo relevance of my prior in vitro findings, and I will present this technique in the next couple of minutes To generate swollen kedia of the environmental mold. Aspergilus fumigatus. Add five milliliters of medium to a six well cell culture plate, then transfer two times 10 to the seventh, resting CEDIA to the plate, and incubate the spores for seven hours at 37 degrees Celsius in 5%carbon dioxide in A BSL two facility.After the swelling period, fluorescently labeled the spores by adding 10 microliters of calcior white stalk solution to each well following gentle mixing. Incubate the plate for an additional 15 minutes under the same conditions to harvest the fluorescently labeled spores, place a 70 micrometer cell strainer on top of a 50 milliliter conical tube. Collect the spores from the plate by pipette, and then pass the media solution through the cell trainer into the tube.Next, spin down the cells at 900 Gs for five minutes at room temperature and discard the media. Then wash the spores with 10 milliliters of PBS under the same centrifuge conditions. Re suspend the spore pellet in two milliliters of PBS and determine the total number of swollen canadia by cell counter spin on the spore suspension again under the same centrifuge conditions as before and afterward, carefully remove the supernatant resus.Suspend the sedimented kedia in a final concentration of one times 10 to the seven cells per 100 microliters of PBS to prepare the mouse for infection with the spore solution, the animal is anesthetized and sedation is confirmed by lack of response to toe pinch. In this video, lys EGFP mice, which carry EGFP under the lysozyme promoter and therefore have eeg FP positive neutrophils are used five minutes after administration of anesthesia and confirmation of sedation using elastic band to suspend an narcotized mouse on a bevel by its teeth under a gooseneck lamp. Use forceps to pull the tongue to one side and insert a 22 gauge indwelling venous catheter into the animal's trachea.After successful insertion of this tubing, use a 100 microliter micro pipette to administer 100 microliters of the spore suspension. Then to enhance the distribution of the fungal particles throughout the lung, mechanically ventilate the infected animal for two minutes with a small animal respirator at a breathing rate of 250 breaths per minute, and an inhalation volume of 300 microliters per breath. Finally, remove the catheter and place the mouse back in its cage for seven hours while the infection progresses.Observe the recovery of the animal every five minutes until ambulatory again, just before the end of the infection period. Melt 20 milliliters of low melting agros in a microwave and keep it warm so that the agros does not solidify before it is injected. Chill a bottle of PBS to four degrees Celsius in the refrigerator at this time as well.At the very end of the infection period, the animal is euthanized. Use needles to fix the mouse to a silicone preparation mat and carefully open the chest using a pair of scissors and forceps to expose the lungs and upper respiratory tract. Starting from the exposed epiglottis, insert another 22 gauge indwelling venous catheter into the trachea.Using a sewing needle, a standard sewing thread is layered around the trachea as preparation for the subsequent agros filling step of the lung. Next, fill a one milliliter syringe with the prewarm aros and administer the solution through the catheter to fill the lungs with it. As soon as the lungs are filled, tie the trachea closed with the prepared sewing thread while removing the catheter and put the whole animal into a refrigerator at four degrees Celsius for 15 minutes.After the aros has solidified, beginning from the trachea, excise the whole lung from the open chest and wash it with the pre-cool PBS using a pair of sharp pointed scissors. Remove the right lung lobe. Briefly dry the bottom of the lobe on a sheet of tissue paper and then fix the lobe to a vibram preparation block using a drop of tissue adhesive for one minute.Next, install the block in the cutting chamber of the vibrato and then fill the chamber with the pre chilled PBS. Set the razor to a mid-range vibration with a medial driving setting and cut a horizontal cross-section of the lung. Then place the upper half of the sliced lung tissue upside down in a five centimeter diameter plastic Petri dish.Fix the lung tissue to the petri dish by placing a flat washer on top of it. Fill the dish with 10 milliliters of PBS and add 10 microliters of cytoxin dye. Finally, install the dish containing the lung tissue under the microscope objective so that the buffer can be warmed by a temperature sensor controlled heater to 37 degrees Celsius for imaging.Different areas along the dissection R scan down to 400 micrometer depth using an illumination wavelength of 800 nanometers detecting green 530 nanometers and red 580 nanometers fluorescence, as well as the second harmonic generation or SHG signal. In the blue councilor fluorescence at 400 to 470 nanometers emission with external non DS scan detectors or NDD shown here in blue is the SHG signal of the alveolar tissue structure of a non-infected lung of a C 57 black six mouse. Note the fibrous tissue at the bottom of the lung slice as compared to the clearly alveolar organization within the breathing active areas at the top of the lung.In this figure, the cyt talk signal of lung cell nuclei cut open during the preparation of the lung slice are visualized in red. This figure shows an overlay of the two channels with a closeup of the boxed area of the tissue shown here.Here. The blue SHG signal indicates the alveolar and fungal structures present in a lung slice of a C 57 black six mouse infected seven hours prior with afu tis.In this figure, the red Cyt talk signal shows not only the nuclei of lung cells cut open during the preparation of the lung slice, but also the DNA nets here. An overlay of the two channels can be seen in this enlarged graphic of the boxed area from the previous figure. Distinct areas of fungal masses, alveoli, and net structures are indicated with the letters FA and N respectively, the combined fungal structure and SHG of the alveolar tissue blue.The cyt talk signal of cell nuclei and nets red, as well as numerous lung infiltrating neutrophils. Green can be visualized here in a slice of lung tissue from a lys EGFP mouse. Seven hours after infection with afu goddess neutrophils in green can be seen migrating and phagocytosing fungal elements in blue in aspers infected lungs as seen by time-lapse.Two photon microscopy of a living lung slice. Seven hours after infection, cell nuclei and net structures are depicted in red. The real time of the experiment is shown on the lower right and the scale bar is indicative of 50 micrometers.Now this method can provide insight into fungal infections. It might also be applied to other systems like viral or bacterial infections, and it might also give insight into the function of other resident immune cells inside the lung.

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Research

JoVE Journal

Immunology and Infection

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 µm including zymosan and IgG-coated beads.

Visualizing the Early Stages of Phagocytosis

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Presented here is a protocol for the induction and analysis of in vitro neutrophil extracellular traps (NETs). Quantification of DNA, cathelicidin (LL37), and enzyme activity yielded data that show the variability in the composition and morphology of NETs induced by microbial and chemical stimuli under similar controlled conditions.

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Learn to visualize neutrophil phagocytosis and NET formation in infected lungs using 2-photon microscopy. This technique enables real-time observation of immune cell dynamics in live tissue.

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

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