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

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)

1. Thaw Cerulean-fluorescent M. marinum glycerol stock from -80 °C and inoculate a 7H10 agar plate with 10% (v/v) OADC, 0.25% glycerol and 50 μg/mL hygromycin. Incubate the plate at 32 °C for around 10 days.
2. Select a colony expressing positive fluorescence and inoculate 3 mL of 7H9 medium with 10% OADC, 0.5% glycerol, and 50 μg/mL hygromycin.
   1. Incubate the inoculation at 32 °C and 100 revolutions per minute (rpm) for 4–6 days until the culture reaches logarithmic phase (OD₆₀₀ = 0.6–1.0).
   2. Subculture (1:100) in 30 mL of fresh 7H9 medium with 10% OADC, 0.5% glycerol, 0.05% Tween-80, and 50 μg/mL until the OD₆₀₀ reaches ~1.0.
      NOTE: For the highest culture quality, a subculture step is recommended at this point. In our experience, adding the clone directly to a large volume of medium will lead to the formation of bacterial clumps.
3. Collect M. marinum cells as described below.
   1. Centrifuge at 3,000 x g for 10 min to collect the M. marinum as a pellet. Discard all but 300 μL of the supernatant and use it for resuspending the pellet.
   2. Add 3 mL of 7H9 medium with 10% glycerol to further resuspend the pellet, then sonicate the suspension in a water bath at 100 W at 15 s ON, 15 s OFF for a total 2 min.
      NOTE: The purpose of sonication is to achieve a single cell homogenate for the inoculum, which will prevent blockage of the microinjection needle.
4. Transfer the bacterial suspension to a 10 mL syringe, then pass through a 5 μm filter to remove any bacterial clumps.
5. Measure the optical density (OD) of the suspension using a spectrophotometer and dilute it to OD₆₀₀ = 1.0 with 7H9 media containing 10% glycerol. Divide the suspension into 10 μL aliquots and store at -80 °C freezer for future use.
6. Confirm the bacterial concentration of the inoculum (cfu/mL) by serial dilution and plating of the bacterial stock on a 7H10 agar plate containing 10% (v/v) of OADC, 0.5% of glycerol, and 50 μg/mL of hygromycin.

2. Zebrafish Embryo Preparation

1. One the day before spawning, set up zebrafish breeding pairs in the breeding chamber.
   NOTE: Add only one pair to each breeding chamber.
2. Collect embryos the next morning within 1 h post fertilization (hpf). Carefully wash the embryos with distilled water and transfer up to 100 embryos into a 100 mm Petri dish containing 30 mL of E3 medium. Incubate at 28.5 °C.
3. After 12 h, observe under a microscope and discard nonfertilized or damaged eggs.
4. At 24 hpf, change the medium to fresh E3 medium with N-phenylthiourea (PTU, 0.2 nM final concentration) to prevent the development of pigment. Incubate the embryos at 28.5 °C until the embryos are ready for microinjection.

3. Infection via Bacterial Microinjection

1. Prepare borosilicate glass microcapillary injection needles as previously described in reference¹³.
2. Zebrafish embryos mounting for infection
   1. Microwave 100 mL of 1% (w/v) and 100 mL of 0.5% (w/v) low melting agarose in an autoclaved E3 medium until the agarose is completely melted. Divide into 1 mL aliquot tubes and store at 4 °C for future use.
   2. Before use, heat the agarose in a 95 °C heating block until it is completely melted. Maintain the agarose in liquid form by placing it in a 45 °C heating block (Figure 2).
   3. Mounting for intramuscular infection in the trunk region
      1. Create the bottom agarose layer by pouring 0.5 mL of 1% (w/v) agarose evenly onto a glass slide. Place on an ice box or cold surface for 3 min to solidify.
      2. Anesthetize the zebrafish embryos (48–72 hpf) in egg water with tricaine (200 µg/mL) and PTU for 5 min prior to mounting. Place up to 60 zebrafish embryos on the bottom agarose layer and lay them out carefully into two rows (Figure 2B).
      3. Remove any remaining water on the bottom agarose layer with tissue paper before adding 0.3 mL of 0.5% (w/v) agarose to create the upper layer. Ensure that the embryos are completely embedded in the agarose. Return the glass slide to the ice box again to solidify the agarose and prevent dehydration.
      4. Keep the top layer of agarose moist by covering the surface with extra E3 egg water.
   4. Mounting for midbrain infection
      1. Cover the groove of a single concavity glass microscopy slide with 1% (w/v) agarose, and then transfer the 4–6 tricaine-anesthetized embryos into the agarose.
      2. Position the head of each embryo upwards carefully with a 10 G needle (Figure 2C).
      3. Once all embryos' positions are fixed, transfer the glass slide to an ice box or cold surface to let the agarose solidify.
         NOTE: Avoid dilution of low melting agarose by minimizing the volume of egg water transfer with the embryos.
3. Bacteria preparation for infection
   1. Add 1 μL of sterile-filtered phenol red (10x) to a 10 μL aliquot of bacterial stock (made in step 1) and mix by vortexing briefly.
      NOTE: The final concentration can be adjusted using sterile PBS.
   2. Sonicate the preparation using 100 W at 10 s ON, 10 s OFF for 1 min to break up any clumps that may have formed¹⁴.
4. Infection via microinjection
   1. Adjust the microinjector and micromanipulator to the proper position and setting for microinjection as previously reported¹³.
   2. Transfer 3 µL of the bacterial preparation using a micro loader into the prepared needle (see step 3.1). Pipette slowly and carefully to avoid forming air bubbles.
   3. For the trunk region infection, inject 100 cfu into the trunk region (Figure 3A). Avoid injecting bacteria into the notochord.
      NOTE: The cfu for injection is estimated by the formula cfu = bacterial stock concentration x dilution factor x injection droplet volume. The actual cfu is confirmed by plating one drop of bacterial inoculum on a 7H10 agar plate containing 10% (v/v) of OADC, 0.5% of glycerol, and 50 μg/mL of hygromycin.
   4. For the midbrain infection, inject about 500 cfu into the midbrain region (Figure 3B).
   5. After microinjection, carefully flush the zebrafish embryos into fresh egg water with a plastic pipette.
      NOTE: Mount embryos in the glass bottom dish as soon as possible to cover the observation of the very early innate immune cell response.

4. Live Imaging of the Infection

1. Fish mounting for live imaging
   1. Transfer up to 10 tricaine-anesthetized embryos to the middle of a glass bottom 35 mm dish. Discard extra E3 medium.
   2. Cover the dish with 1% low melting point agarose and orient the zebrafish embryos carefully using a 10 G needle. Incubate the glass bottom dish on ice for 10 s to solidify the agarose.
      NOTE: For midbrain injection, embryos should be mounted in the agarose with the head directed upwards (Figure 4A). For the trunk region intramuscular infection, embryos should be mounted laterally in the agarose (Figure 4B).
   3. Once it has completely solidified, cover the agarose with a layer of egg water (plus 1 x tricaine and PTU).
2. Three-color high-resolution time lapse confocal microscopy
   NOTE: The following steps are operated on a confocal microscopy equipped with a 63.0x 1.40 oil UV objective lens.
   1. Set the temperature of the environmental chamber to 28.5 °C. Place some wet tissue paper inside the chamber to provide humidity and prevent evaporation of the egg water (Supplemental Figure 1).
   2. Place the 35 mm glass bottom dish with the zebrafish in the environmental chamber.
   3. Open the confocal software and initialize the stage. Switch to the 63.0 x 1.40 oil UV objective, and locate the zebrafish using the bright field channel with a differential interference contrast (DIC) filter.
   4. Open the 405 Diode, Argon (20% power), and DPSS 561 nm laser. Set up the appropriate laser power and spectrum settings.
      NOTE: The following are the spectrum settings for Cerulean (excitation = 405 nm; emission = ~456–499 nm), eGFP (excitation = 488 nm; emission = ~500–550 nm), DsRed2 (excitation = 561 nm; emission = ~575–645 nm) (Supplemental Figure 2B).
   5. Choose the "XYZ" "Sequential Scan" acquisition mode and set images format to "512 x 512 pixels" (Supplemental Figure 2A).
   6. 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. A "Pause" can be added at the end of the program (Supplemental Figure 2C).
   7. Define the loop and cycle of the program.
   8. Save the file.

5. Single Cell UV Irradiation to Induce Apoptosis and Live Imaging

1. Mount fish as described in step 4.1.
2. Imaging the midbrain region of 3 days post fertilization (dpf) macrophage specific transgenic Tg(mfap4-eGFP) embryo¹⁵
3. Select the region of interest of one single fluorescently labeled macrophage and scan at 400 Hz speed and 6% UV laser power for 50 s.
   NOTE: Scanning speed and time should be optimized based on the individual microscope. Scanning time should be optimized to cause the extensive DNA damage that will subsequently cause target cell apoptosis, but not photobleach the entire cell.
4. Repeat the above step to irradiate more target cells.
5. Perform time lapse imaging of the midbrain region as described in section 4.

6. Image Processing

1. Perform "Maximum Projection" for the acquired images.
2. Find and mark the XY position and time of the target cells under the "Maximum Projection" view.
3. Go back to the standard view to find and mark the Z position of the target cells.
4. Crop the single layer image of the target cells.
5. Export the overlay channel and bright field as videos in AVI format.
6. Crop the area of interest of the overlay channel and bright field in ImageJ.
7. Combine the two videos in the last step vertically and save as one AVI format video in ImageJ.