The overall goal of this procedure is to visualize non litic exocytosis of cryptococcus neoformans from primary bone marrow neuron macrophages. This is accomplished by first plating and infecting activated neuro macrophages with fungal cells to allow for phagocytosis and internalization of Xeomin. The second step is to remove extracellular fungal cells to ensure a clear monolayer of infected and uninfected macrophages.
Next macrophages are incubated in a carbon dioxide temperature controlled chamber and recorded for 24 hours in order to capture instances of non litic exocytosis and other interactions. The final step is to create a movie by compressing all the frames made in a 24 hour period. Ultimately, macrophage visualization with digital light microscopy is used to observe the variety of non litic exocytosis types that macrophages undergo throughout a 24 hour infection.
This method can help answer key questions in the host pathogen interaction field, such as timing and quantification of cellular events On the day prior to the experiment, select one individual senior foreman's colony that was grown on a sbarro agar plate and inoculate. 10 milliliters of sbarro broth. Allow the culture to grow overnight at 37 degrees Celsius, shaking at a speed of 250 RPM following euthanasia of mice as described in the written protocol.
Sterilize the entire body with 70%ethyl alcohol. Remove both femurs and tibia and place the bones in sterile duo's, modified eagle medium, or DMEM. Then remove excess tissue and muscle using delicate wipes until the bones are completely clean.
Place only intact bones in a Petri dish containing 70%ethyl alcohol and incubate for three minutes to kill associated microorganisms. Remove the bones and place in a Petri dish with sterile DMEM. Next, carefully cut the ends of the bones.
Hold the bone over an MT 50 milliliter conical tube placed on ice, and use a 25 gauge needle to carefully flush 10 milliliters of cold DMEM through each bone. Then centrifuge the collected cell suspension at room temperature for 10 minutes at 650 times G.During this centrifugation, prepare and filter sterilize bone marrow macrophage feeding media as described in the text protocol. Next, resuspend the resulting palette with 10 milliliters of feeding media.
Pass the cell suspension through a 70 micrometer cell strainer to disrupt cell clumps and remove large debris. Then plate one milliliter of the strain cell suspension into 10 milliliters of feeding media, Petri dishes that are specified for tissue culture use. Allow the cells to adhere at 37 degrees Celsius with 10%carbon dioxide for seven days.
Changing the feeding media as described in the text protocol the day prior to the experiment. Detached macrophages from the plate by removing feeding media and add five milliliters of a gentle cell stripping reagent to the plate following incubation of the cells for five to 10 minutes. At 37 degrees Celsius, remove the macrophages with gentle pipetting.
Pellet the cells by centrifugation at 650 times G for 10 minutes before reus bending the pellet in one milliliter of feeding media. Using a one to 20 dilution, calculate the concentration of macrophages by pipetting 10 microliters of the cell suspension into a hemo cytometer using 14 millimeter glass bottomed. Petri dishes place a concentration of one times 10 to the fifth macrophages directly into the inner well, which holds a maximum of 200 microliters.
Take care not to exceed this volume. Allow the cells to adhere to the glass Petri dish by placing the dishes in a 37 degrees Celsius incubator for one hour. Then add one to two milliliters of feeding media supplemented with lipopolysaccharide at one milligram per milliliter and interferon gamma.
At 500 units per milliliter, allow the cells to incubate overnight on the day of the experiment. Remove one milliliter of an overnight inoculated G cell culture and pal the yeast cells by centrifugation for five minutes of 420 times G.To remove media and extracellular cryptococcal products, wash the cells three times with phosphate buffered saline or PBS followed by centrifugation as before. After resus suspending the yeast cell pellet in one milliliter of sterile PBS and making a one to 100 dilution pipette 10 microliters of the dilution into a hemo cytometer and count the cells.
Calculate the concentration of yeast cells needed for a multiplicity of infection of one to five. Add the calculated volume of cryptococcal cell suspension to one milliliter of feeding media with the monoclonal antibody, 18 B seven at a concentration of 10 micrograms per milliliter. Incubate the cell suspension at room temperature for five minutes.
Next, remove the feeding media from the glass Petri dish and wash the macrophages once with sterile PBS. Then add 100 microliters of opsonize cmin directly to the well, followed by incubation with the yeast for two hours at 37 degrees Celsius and 10%carbon dioxide side. After co incubation checked by microscope that the macrophages have internalized c eForms to be considered internalized cryptococcal cells should be clearly seen within the confines of the macrophage as indicated by the large arrow head.
The smaller arrow head indicates an uninfected macrophage with no fungal cells. Wash the Petri dish three times with sterile PBS to remove any and all extracellular c neoformans. Then add one to two milliliters of feeding media without the monoclonal antibody to perform these experiments.
A microscope equipped with an attached incubating chamber that includes carbon dioxide delivery and a heating unit is required prior to the experiment. Prewarm the incubating chamber for at least 30 minutes at 37 degrees Celsius. Ensure that the carbon dioxide unit reads 5%at the start of the experiment.
Place the glass bottomed Petri dish on the microscope platform cover with a carbon dioxide lit and close all doors to ensure no heat escapes using the 10 x objective with the microscope in phase contrast, focus on a clear field of infected macrophages set up the microscope software to take an image every four minutes for a 24 hour period. After 24 hours, the experiment will have reached completion and a total of 361 images will have been collected. Export these images as JPEGs at a 5%compression.
Using image J software view the images as a visual stack at five frames per second. The initial frame of the movie shows many macrophages throughout the field with some that are uninfected and some that have sized varying amounts of cryptococcal cells. As the movie progresses, the cells are all mo and can be seen moving about the field in this movie, it is clear that the cryptococcal cells are within the confines of an infected macrophage.
The macrophage becomes agitated and a yeast cell can be seen, but in within the FGA zone, cryptococcal cells are then quickly expunged into the surrounding environment. A second infected macrophage undergoes non litic exocytosis and more yeast cells are released into the extracellular space. The host cells remain viable after cryptococcal exocytosis as indicated by their ability to continue moving.
On occasion, macrophages will interact in such a way that facilitates transfer cryptococcal cells between host cells. The yeast cells are not exposed to the extracellular environment and the host cells remain viable. In this movie, two infected macrophages create cell to cell bridges on two separate occasions to shuttle cryptococcal cells from one macrophage to another.
After watching this video, you should have a good understanding of how to utilize digital light microscopy to visualize macrophages undergoing non lytic exocytosis, and other host pathogen interactions.
