Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The overall goal of this procedure is to compare the ability of macrophages to control the growth of ceto fungal candia ex vivo. This is accomplished by first labeling the alveolar macrophages in vivo with a lipophilic dye PKH 26. The second step of the procedure is to harvest alveolar macrophages from the inoculated mice by bronchoalveolar lavage.

The third step is to culture and collect GFP positive fungal spores. The final steps are to challenge macrophages with GFP positive candia and fixed cells at predetermined time points. Ultimately, with minimal ex vivo cell manipulation, confocal microscopy can reveal the ability of alveolar macrophages to control the growth of phagocytose.

Spores First dilute the lipophilic dye P HK 26, 1 to five. In Diluent C, the dye will be taken up by the FOC acidic cells. Load 100 microliters of diluted P HK 26 into a one half cc syringe and inject the dye intravenously retroorbital, or by the tail vein.

Estimate that each mouse will provide three to 500, 000 labeled macrophages after 10 days. Euthanize the mice and prepare to open the chest cavity from the diaphragm. Cut a small hole in the rib cage to the right of the thorax to deflate the lungs and for later viewing of the lungs inflation.

Next, locate and isolate the trachea from the surrounding tissues. Then using curved forceps, carefully cut away the adventitia, which covered the trachea. Once exposed, insert a catheter into the trachea.

It is very important to avoid puncturing the trachea. In executing this maneuver, secure the catheter with a 10 centimeter length of suture looped around the trachea. Then remove the needle from the catheter.

Next, fill a six milliliter syringe with lavage fluid and attach it to a four-way stop cock. Attach an empty six milliliter syringe at another position and carefully connect the open port to the catheter. With the assembly in place, open the stop cock to the full syringe and slowly inject about one milliliter of solution into the lungs.

Next, turn the stop cock to close the syringe and to withdraw fluid. Then carefully withdraw the macrophage rich lung fluid while observing the lungs deflate. Repeat this process until all the solution has been injected into and collected from the lungs.

Expect some fluid loss. You'll not be able to remove the fluid from the lungs if the catheter is in the wrong position. Carefully move the catheter back and forth until the fluid can easily be withdrawn from the lungs.

Be certain not to allow the catheter to fall out. Then transfer the collected lavage fluid into a 50 milliliter conical to collect the cells centrifuge the fluid at 300 Gs for five minutes. At room temperature, pour off the supernatant and if desired, save it for cytokine analysis.

Resuspend the cells in one to five milliliters of RPMI 1640 with 10%FBS and determine their density. At least 95%of the cells should be macrophages to culture. Aspergillus fumigatus expressing GFP Berran brain heart infusion.

A agri slants treated with 0.05 grams of chloramphenicol and 0.5 grams of gentamycin per liter. Incubate the fungus on the slants loosely capped in the dark and at room temperature. After seven to 10 days, harvest the canadia by washing each slant with 10 milliliters of 0.01%Tween 20 in DPBS use a STR PET to loosen the fungus and use several rinses to increase the yield.

Pass the suspension through a 100 micron strainer and collect the filtrate containing the kedia. In a 50 milliliter conical pool the kedia with a 10 minute spin at 400 Gs at room temperature. Then resuspend them in 50 milliliters of DPBS and determine their concentration with a hemo cytometer.

Lastly, wash the kedia twice more with DBBS and resuspend them at the desired concentration. First cover slips and plates must be prepared under a biological safety cabinet. Soak 22 millimeter square cover slips in 70%ethanol for five to 10 minutes.

Then rinse them off in PBS for each time point to process, use forceps to gently place single sterilized cover slips into the wells of a six well plate load a well for each condition tested onto each cover slip seed about 1 million PKH 26 labeled macrophages in 300 microliters of RPMI 1640 with 10%FBS incubate the cells at 37 degrees Celsius with 5%carbon dioxide overnight. The following morning to each seated cover slip at 10 million GFP expressing aspergillus fuma Gattis kedia in 100 microliters of warm RPMI 1640 with 10%FBS. Return the plates to the incubator for the duration of the challenge and fix the cells at predetermined time intervals to score the results.

To fix a plate of cells, add one volume of 2%formaldehyde to each well, and let the plate sit for two hours at room temperature. After two hours, move the plate to four degrees Celsius until the other plates have also been fixed. First, prepare a glass slide with six microliters of fluorescent mounting medium with dappy When all the plates are fixed, gently remove the cover slips with forceps and rinse them in a bath of DPBS for a minute.

Then blot their edges to remove excess fluid. Next, gently lay the cover, slip down into the mounting medium prepared on the glass slide earlier. There is no need to press the slide for the medium to spread.

Seal the slide with nail polish and allow it to air dry. Store the slides at room temperature in the dark until they can be viewed in vivo. Labeled alveolar.

Macrophages were collected and then exposed to a fungal challenge. Wild type mice and mice carrying a mutation in N-A-D-P-H. Oxidase were used to harvest macrophages after three or seven hours.

Phagocytosis was similar in the wild type and mutant strains. Cells containing candia positive for both PKH 26 and GFP were abundant in all three genotypes, however, at 14 hours differences emerged with respect to the ability of macrophages to control the growth of phagocytosed Candia. Many intact wild-type macrophages containing phagocytosed candia were found.

This was also observed in cells with a functional N-A-D-P-H oxidase enzyme in the NCF one. Background by contrast, intact alveolar macrophages lacking N-A-D-P-H oxidase were scarce, suggesting that Phace toast Kedia had grown destroying the cells. This also suggests that controlling the growth of phace toast Kedia is N-A-D-P-H oxidase dependent Once mastered, this lavage technique can be done in about five minutes per mouse if performed properly.