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

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.