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

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.