Name: visualization of macrophage lytic cell death during mycobacterial infection in zebrafish embryos via intravital microscopy
Uploaded: 2019-01-09T15:00:00
Description: Watch this Scientific Journal Video about Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy at JoVE.com

We thank Dr. Zilong Wen for sharing zebrafish strains, Dr. Stefan Oehlers and Dr. David Tobin for sharing M. marinum related resources, Yuepeng He for assistance in figure preparation. This work was supported by the National Natural Science Foundation of China (81801977) (B.Y.), the Outstanding Youth Training Program of Shanghai Municipal Health Commission (2018YQ54) (B.Y.), Shanghai Sailing Program (18YF1420400) (B.Y.), and Open Fund of Shanghai Key Laboratory of Tuberculosis (2018KF02) (B.Y.).

Zebrafish were raised under standard conditions in compliance with laboratory animal guidelines for ethical review of animal welfare (GB/T 35823-2018). All zebrafish experiments in this study were approved (2019-A016-01) and conducted at Shanghai Public Health Clinical Center, Fudan University.
1. M. marinum Single Cell Inoculum Preparation (Figure 1)
<ol>
	<li>Thaw Cerulean-fluorescent M. marinum glycerol stock from -80 &#176;C and inoculate a...

<ol>
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This protocol describes the visualization of macrophage death during mycobacterial infection. Based on factors such as the integrity of the cell membrane, infection driven cell death can be divided into apoptosis and lytic cell death24,25. Lytic cell death is more stressful for the organism than apoptosis, because it triggers a strong inflammatory response 24,25. Observation of lytic cell death in vivo is...

Mycobacterial infection has been demonstrated to cause host immune cell death1. For example, an attenuated strain will trigger apoptosis in macrophages and contain the infection. However, a virulent strain will trigger lytic cell death, causing bacterial dissemination1,2. Considering the impact these different types of cell death have on host anti-mycobacterial response, a detailed observation of macrophage cell death during mycobacterial infection in vivo is needed.
The conventional methods for measuring cell death are to use dead cell stains, such as Annnexin V, TUNEL, or acridine orange/propidium iodide staining3,4,5. However, these methods are unable to shed light on the dynamic process of cell death in vivo. The observation of cell death in vitro has already been facilitated by live imaging6. However, whether the results accurately mimic physiological conditions remains unclear.
Zebrafish have been an excellent model for studying host anti-mycobacterium responses. It has a highly conserved immune system similar to that of humans, an easily manipulated genome, and the early embryos are transparent, which allows for live imaging7,8,9. After infection with M. marinum, adult zebrafish form typical mature granuloma structures, and embryonic zebrafish form early granuloma like structures9,10. The dynamic process of innate immune cell-bacteria interaction has been explored previously in the zebrafish M. marinum infection model11,12. However, due to high spatial-temporal resolution requirement, the details surrounding the death of the innate immune cells remain largely undefined.
Here we describe how to visualize the process of macrophage lytic cell death triggered by mycobacterial infection in vivo. This protocol may also be applied to visualizing cellular behavior in vivo during development and inflammation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Tween-80</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringe</td>
			<td>Solarbio</td>
			<td>YA0552</td>
			<td></td>
		</tr>
		<tr>
			<td>10% OADC</td>
			<td>BD</td>
			<td>211886</td>
			<td></td>
		</tr>
		<tr>
			<td>3-aminobenzoic acid</td>
			<td>Sigma</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>5 &#956;m filter</td>
			<td>Mille X</td>
			<td>SLSV025LS</td>
			<td></td>
		</tr>
		<tr>
			<td>50 &#956;l/ml hygromycin</td>
			<td>Sangon Biotech</td>
			<td>A600230</td>
			<td></td>
		</tr>
		<tr>
			<td>7H10</td>
			<td>BD</td>
			<td>262710</td>
			<td></td>
		</tr>
		<tr>
			<td>7H9</td>
			<td>BD</td>
			<td>262310</td>
			<td></td>
		</tr>
		<tr>
			<td>A glass bottom 35 mm dish</td>
			<td>In Vitro Scientific</td>
			<td>D35-10-0-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sangon Biotech</td>
			<td>A60015</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>TCS SP5 II</td>
			<td></td>
		</tr>
		<tr>
			<td>Enviromental Chamber</td>
			<td>Pecon</td>
			<td>temp control 37-2 digital</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microloader</td>
			<td>Eppendorf</td>
			<td>No.5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slide</td>
			<td>Bioland Scientific LLC</td>
			<td>7105P</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sangon Biotech</td>
			<td>A100854</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Keelrein</td>
			<td>PH-140(A)</td>
			<td></td>
		</tr>
		<tr>
			<td>M.marinum</td>
			<td></td>
			<td></td>
			<td>ATCC BAA-535</td>
		</tr>
		<tr>
			<td>Microinjection needle</td>
			<td>World Precision Instruments</td>
			<td>IB100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Eppendorf</td>
			<td>Femtojet</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>NARISHIGE</td>
			<td>MN-151</td>
			<td></td>
		</tr>
		<tr>
			<td>msp12:cerulean</td>
			<td></td>
			<td></td>
			<td>Ref.: PMID 25470057; 27760340</td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>Sigma</td>
			<td>P3532</td>
			<td></td>
		</tr>
		<tr>
			<td>PTU</td>
			<td>Sigma</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>Single concavity glass microscope slide</td>
			<td>Sail Brand</td>
			<td>7103</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>SCICNTZ</td>
			<td>JY92-IIDN</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer (OD600)</td>
			<td>Eppendorf AG</td>
			<td>22331 Hamburg</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>OLYMPUS</td>
			<td>SZX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tg(mfap4:eGFP)</td>
			<td></td>
			<td></td>
			<td>Ref.: PMID 30742890</td>
		</tr>
		<tr>
			<td>Tg(coro1a:eGFP;lyzDsRed2)</td>
			<td></td>
			<td></td>
			<td>Ref.: PMID 31278008</td>
		</tr>
		<tr>
			<td>Tg(mpeg1:LRLG;lyz:eGFP)</td>
			<td></td>
			<td></td>
			<td>Ref.: PMID 27424497; 17477879</td>
		</tr>
	</tbody>
</table>

visualization of macrophage lytic cell death during mycobacterial infection in zebrafish embryos via intravital microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

Mycobacterium infection can trigger different host responses based on the routes of infection. In this protocol, zebrafish embryos are infected by intramuscular microinjection of fluorescently labeled bacteria into the midbrain or trunk (Figure 3) and observed by confocal live imaging. Infection via these two routes will locally restrict the infection causing innate immune cell recruitment and subsequent cell death.
Visualizing the details of innate immune cell de...

Watch this Scientific Journal Video about Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy at JoVE.com

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

This protocol can clearly differentiate macrophage cell death modes during mycobacterial infection. By observing multiple embryos in parallel, it greatly increases the probability of capturing the entire macrophage lytic cell death process. This protocol may also be applied to the observation of cell death and other cellular behavior in similar scenarios involving infection or sterile inflammation.During the extended live imaging, it is very important to keep the intensity of the laser as low as possible to avoid photobleaching and toxicity. After inoculation according to the manuscript, centrifuge the culture at 3000 times G for 10 minutes to collect the Mycobacterium marinum as a pellet. Discard all but 300 microliters of the supernatant, and re-suspend the pellet.Add three milliliters of 7H9 medium with 10%glycerol to further re-suspend the pellet, and then sonicate the suspension in a water bath at 100 watts, with 15 seconds on and 15 seconds off, for a total of two minutes to achieve a single cell homogenate. Transfer the bacterial suspension to a 10 milliliter syringe, and pass through a five micron filter to remove any bacterial clumps. Using a spectrophotometer, measure the optical density of the suspension, and dilute it with 7H9 media containing 10%glycerol to OD 600 at one.Divide the suspension into 10 microliter aliquots, and store at negative 80 degrees Celsius freezer for further use. First, heat the agarose in a 95 degree Celsius heating block until it is completely melted. Maintain the agarose in liquid form by placing it in a 45 degrees Celsius heating block.To mount for intramuscular infection in the trunk region, create the bottom agarose layer by pouring 0.5 milliliters of 1%weight by volume agarose evenly onto a glass slide. Place the slide on an icebox or cold surface for three minutes to solidify. After anesthetizing the zebrafish embryos, place up to 60 zebrafish embryos on the bottom agarose layer, and lay them out carefully in two rows.Remove any remaining water on the bottom agarose layer with tissue paper, before adding 0.3 milliliters of 0.5%weight by volume agarose to create the upper layer. Ensure that the embryos are completely embedded in the agarose. Return the glass slide to the icebox again to solidify the agarose.Keep the top layer of the agarose moist by covering the surface with extra E3 egg water. Next, adjust the microinjector and micromanipulator to the proper position and setting for microinjection. Transfer three microliters of prepared bacterial culture into the prepared needle using a microloader.Pipette slowly and carefully to avoid forming air bubbles. Inject 100 CFU into the trunk region. After microinjection, carefully flush the zebra fish embryos into fresh egg water with a plastic pipette.To mount for the midbrain infection, use a plastic pipette to transfer the four to six tricaine-anesthetized embryos into the agarose. Position the head of each embryo upwards carefully with a 10-gauge needle. Once all embryos positions are fixed, transfer the glass slide to an icebox or cold surface to let the agarose solidify.Inject 500 CFU into the midbrain. After microinjection, carefully flush the zebrafish embryos into fresh egg water with a plastic pipette. Once the agarose has completely solidified, cover the agarose with a layer of egg water.After setting up the environmental chamber, place the 35 millimeter glass bottom dish with the zebrafish in the environmental chamber. Open the 405 diode, argon at 20%power, and DPSS 561 nanometer laser. Set up the appropriate laser power in spectrum settings.Choose the XYZ sequential scan acquisition mode, and set images format to 512 by 512 pixels. Switch to live data mode, target the position of the first zebrafish, and mark the begin and end Z position. Repeat this process for each of the remaining embryos.Add a pause at the end of the program. Define the loop and cycle of the program, and save the file. In this study, we utilized previously reported transgenic coro1a:eGFP;lyzDsRed2, and transgenic mpeg1loxP;DsRed:loxPeGFP;lyz:eGFP, to distinguish the macrophages and the neutrophils in vivo.A macrophage heavily engorged with bacteria became round and displayed reduced motility, with eventual cytoplasmic swelling, rupturing of the cell membrane, and quick dissemination of the cytoplasmic content. UV-irradiated macrophages showed typical apoptotic cell phenotypes such as cell shrinkage, nuclear fragmentation, and chromatin condensation. It was also observed the macrophages actively phagocytosed and disseminated M.merinum.However, neutrophils had limited phagocytic capability, and quickly underwent lytic cell death without obvious bacterial engorgement. Combined with powerful gene editing tools, this protocol can provide an effective platform for further understanding the effect of a variety of factors on host-pathogen interaction in vivo.

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Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated ...