This technique can help answer key questions in the cellular infection and microbiology field, such as how hemocytes contribute to the overall immune response in fruit flies. The main advantage of this technique is that it can be performed in the laboratory, using previously infected larvae. Alternatively, hemocytes from uninfected larvae can be used for ex vivo infection.
Visual demonstration of this method is critical because the imaging steps are difficult to learn because of the complexities of the confocal microscope and the options for 3D model reconstruction. To begin the protocol, layer two to three pieces of ten centimeter by ten centimeter paraffin film under a stereo microscope. Next, prepare the glass capillary by setting the capillary polar heater to 55%of maximum.
Pole the capillary tube to a sharp point of approximately ten micrometers. Backfill the capillary with mineral oil. Assemble the filled capillary tube onto the nanoinjector.
And open the fused capillary tube tip by breaking off the tip with forceps. Eject as much oil as possible from the capillary tube tip, then fill the tube with drosophila hemocyte isolating medium, or DHIM. For easy visualization of the border of hemolymphan oil, include an air bubble between the oil and DHIM.
Using forceps, gently pick ten third instar drosophila larvae from the inside wall of food vials, and place them into a 100 micrometer strainer. Pour five milliliters of sterile water onto the larvae, and shake the strainer for five seconds. Place the strainer onto a task wipe to remove the excess water.
Transfer the larvae into a 1.5 milliliter microcentrifuge tube, anesthetize them with CO2 gas for five seconds. Then, place the larvae onto paraffin film under the stereo microscope, with the dorsal side facing up. Place the glass capillary lightly onto the larval posterior body to hold it in place, and disrupt posterior cuticle open using fine-pointed forceps.
Allow the hemolymph to flow onto the paraffin film. Make a pool of hemolymph, including hemocytes, from 20 to 50 larvae at a time, on the paraffin film. Use the glass capillary on the nanoinjector to take up the pooled hemolymph.
Eject the hemolymph into a 1.5 milliliter microcentrifuge tube containing 500 microliters of DHIM. Place yeast paste on a grape juice agar plate. Make a fine cut in the agar where the larvae can migrate to avoid drying.
Assemble a 0.001 millimeter pointed Tungsten needle with holding forceps using paraffin film. Pipette 50 microliters of high titer, mCherry-expressing C.Burnetii onto the paraffin film under the stereo microscope, and place the larvae into the pool of bacteria. Prick the larvae with a Tungsten needle.
Transfer the larvae onto an agar plate. Transfer the remaining pathogen medium onto the agar plate, and seal the plate with paraffin film. Keep the larvae on the plate in moist air until the desired time post-infection.
Leave the C.Burnetii-infected larvae on the plate for 24 hours. Place a 12 millimeter round cover glass in a well of a 24-well plate. Pipette 500 microliters of DHIM into the well.
Extract and take up hemolymph from previously-infected larvae, then eject the hemolymph into the well. Centrifuge the plate at 1000 G for five minutes. Configure the confocal microscope for three-color imaging of DAPI, EGFP, and mCherry.
Place the sample on the microscope and focus on the sample using a 63x 1.4 numerical aperture objective. Locate desired hemocytes in the field of view for imaging. Then, adjust the laser power and detector gains to achieve the appropriate exposure of the sample.
Check multiple Z planes to ensure the exposure level is appropriate for the entire sample thickness. Next, find the top and bottom position on the Z axis of a whole hemocyte. Set these positions as the start and end positions for Z sectioning.
Use scanning zoom to image the area containing the hemocyte. Collect the image series at the appropriate resolution, such as 1024 by 1024 pixels in the X Y plane, and 0.3 micrometer spacing in the Z dimension. Import the Z sectioned image series file into the software associated with the confocal microscope for 3D model reconstruction.
Select a cell-showing co-localization of nuclei stained with DAPI and mCherry-expressing C.Burnetii in an EGFP-expressing hemocyte. Crop the image series to contain only the single cell. Select the 3D viewer option, which reconstructs the 3D model using the software's pre-packaged algorithm.
Choose the desired imaging type for 3D representation among blend, surface, and mixed options. In this method, hemocytes, nuclei, and C.Burnetii are shown using surface models. Observe the 3D reconstructed cell from various viewing positions by holding the mouse button and dragging the cursor around the screen.
Adjust the cell orientation and position of the modeled light source to optimize the image. Finally, take cross sections through the model using clipping and sectioning commands to visualize the interior contents of the hemocyte. Visualization of the extracted living hemocytes expressing EGFP under control of the hemolectin driver from multiple images show that most DAPI-positive cells are also EGFP-positive.
Cross sections of Coxiella Burnetii-infected hemocytes confirm the presence of mCherry-expressing bacteria in the interior of the cell. Images were reconstructed into 3D models and we observed that in vivo infected hemocytes exhibited greater cytoplasmic extensions and were flatter in nature. While ex vivo infected hemocytes were more spherical.
Cross sections of either model confirm the presence of mCherry-expressing Coxiella in the interior of the cell. Extracted hemocytes were infected with IIV6 and applied to qRT PCR analysis, which showed significantly higher levels of the IIV6-193R transcript between mock and IIV6-infected cells. While attempting this procedure, it's important to remember to have all media prepared ahead of time and equilibrated to room temperature so that cells are not without the proper nutrients for too long.
Following this procedure, other methods, like single-cell RNA seek, can be performed to answer additional questions, like the overall host response to infection. After its development, this technique helps researchers in immunology and microbiology explore pathogen invasion and host responses in fruit flies, or other animals with open circulatory systems.
