Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

All animal work was done in accordance with regulations and guidelines of the Institute for Animal Studies at Albert Einstein College of Medicine.

1. Growth Conditions of C. neoformans H99 (Serotype A)

1. Grow individual C. neoformans (Cn) colonies on Sabouraud agar plates.
2. One day prior to the experiment, select one colony and inoculate 10 ml of Sabouraud broth.
3. Allow culture to grow overnight at 37 °C shaking at a speed of 250 rpm.

2. Isolation and Preparation of Primary Bone Macrophages from C57/Bl6 Mice

1. Euthanize mice by placing animal in a CO₂ chamber until no longer breathing. As a second measure, cervically dislocate the neck before beginning dissection. Sterilize entire body with 70% ethyl alcohol.
2. Remove both femurs and tibia and place bones in sterile DMEM.
3. Remove excess tissue and muscle using delicate wipes until bones are completely clean.
4. Place only intact bones in a Petri dish containing 70% ethyl alcohol and incubate for 3 min to kill associated microorganisms. Remove bones and place in Petri dish with sterile DMEM.
5. Carefully cut ends of bones. Hold bone over an empty 50 ml conical tube placed on ice and carefully flush 10 ml of cold DMEM using a 25 G needle through each bone.
   NOTE: Each mouse will yield 4 bones (2 femurs, 2 tibias). Therefore one mouse should yield approximately 40 ml of cell suspension.
6. Centrifuge cell suspension at room temperature for 10 min at 650 x g.
7. During this centrifugation, prepare and filter sterilize bone marrow macrophage feeding media which consists of DMEM supplemented with 20% L929, 10% fetal calf serum, 10% NCTC-109, 1% Pen-Strep, 1% HEPES, 1% L-Glutamine, 1% non-essential amino acids, 0.1% 2-Mercaptoethanol.
8. Resuspend resulting pellet with 10 ml of feeding media. Pass cell suspension through a 70 µm cell strainer to disrupt cell clumps and remove large debris.
9. Plate 1 ml of cell suspension into 10 ml of feeding media in Petri dishes that are specified for tissue culture use (for a total of 10 plates).
10. Allow cells to adhere at 37 °C with 10% CO₂. Add 5 ml of fresh feeding media on day 3. On day 7, completely replace the feeding media and macrophages are ready to be used.
11. The day prior to the experiment, detach macrophages from plate by removing feeding media and add 5ml of a gentle cell stripping reagent to plate.
12. Incubate for 5-10 min at 37 °C and remove macrophages with gentle pipetting.
13. Pellet cells by centrifugation at 650 x g for 10 min.
14. Resuspend pellet in 1 ml of feeding media. Using a 1:20 dilution, calculate the concentration of macrophages by pipetting 10 µl of cell suspension onto a haemocytometer.
15. Using 14 mm glass bottomed Petri dishes, plate a concentration of 1 x 10⁵ macrophages directly onto the inner well which holds a maximum of 200 µl, so take caution to not exceed this volume as shown in Figure 4.
16. Allow the cells to adhere to the glass Petri dish by placing the dishes in a 37 °C incubator for 1 hr.
17. Add 1-2 ml of feeding media supplemented with LPS at 1 mg/ml and IFNg at 500 u/ml and allow cells to incubate overnight.

3. Co-incubation of Primary Murine Macrophages and C. neoformans

1. On the day of experiment, remove 1 ml of overnight inoculated culture and pellet yeast cells by centrifugation for 5 min at 420 x g.
2. Wash cells 3 times with sterile PBS to remove media and extracellular cryptococcal products like the polysaccharide glucuronoxylomannan (GXM) at the speed and time mentioned above.
3. Resuspend cell pellet in 1 ml of sterile PBS and make a 1:100 dilution. Pipette 10 μl of the dilution onto a haemocytometer and count cells. Add yeast cells at a MOI of 1:5. For example, if there are 1 x 10⁵ macrophages/well, make a cell suspension of 5 x 10⁶ /ml of Cn and add 100 µl of this solution for a total amount of 5 x 10⁵ Cn.
4. Add calculated volume of cryptococcal cell suspension to 1 ml of feeding media with the mAb 18B7 antibody at a concentration of 10 μg/ml. Incubate cell suspension at room temperature for 5 min.
5. Remove feeding media from glass bottomed Petri dish and wash 1x with sterile PBS. Add 100 μl of opsonized Cn directly to well.
6. Allow cells to incubate for 2 hr at 37 °C with 10% CO₂.
7. After co-incubation, check by microscope that macrophages have internalized Cn. To be considered internalized, cryptococcal cells should clearly be seen within the confines of the macrophage.
8. Wash Petri dish 3x with sterile PBS to remove any and all extracellular Cn.
9. Add 1-2 ml of feeding media without the addition of mAb 18B7, and set up microscope.

4. Visualizing Non-lytic Exocytosis in Real-time Utilizing Digital Light Microscopy

NOTE: To perform these experiments, a microscope that comes with an attached incubating chamber that includes CO₂ delivery and a heating unit is required.

1. Prior to the experiment, pre-warm the incubating chamber for at least 30 min to 37 °C. Ensure that the CO₂ unit reads 5% at the start of the experiment.
2. Place glass bottomed Petri dish on microscope platform, cover with CO₂ lid, and close all doors to ensure no heat escapes.
3. Using the 10X objective with the microscope in phase contrast, focus on a clear field of infected macrophages. Based on the experiment, determine the objective used by how many macrophages need to be studied.
4. Set up microscope software to take an image every 4 min for a 24 hr period.
5. After 24 hr, 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 images as a Visual Stack at 5 frames per sec. If the movie needs to be viewed for publication or a presentation, save it as either an .avi or .mov file depending on which format works best for the viewer.