The Effect of Aspirin and Ibuprofen on the Phagocytic Ability of Macrophages

1. Safety considerations

1. Personnel should use personal protective equipment (PPE).
2. Handle the pathogen and macrophages in a Class II Biological safety cabinet.

2. Preparation of macrophages

1. Cultivate a murine macrophage cell line, RAW 264.7, in a tissue flask containing 10 mL of Roswell Park Memorial Institute-1640 (RPMI-1640) medium, supplemented with 20 mg/mL streptomycin, 2 mM L-glutamine, 20 U/mL penicillin and 10% fetal bovine serum (FBS).
2. Place the flask in a 5% carbon dioxide (CO₂) incubator set at 37˚C until 80% confluence is achieved.
3. Gently scrape off the cells using a cell scraper.
4. Transfer the suspended cells into a 15 mL centrifuge tube.
5. Use the trypan blue stain to determine cell viability. 
   NOTE: A viability score that is above 85% is preferred.
   Use a hemocytometer to adjust the cell concentration of macrophages to 1 × 10⁵     cells/mL using a 10 mL solution of fresh, sterile RPMI-1640 medium. Dispense 100 µL suspension of cells into wells of a sterile 96-well flat-bottom microtiter plate. Incubate the plate overnight in a 5% CO₂ incubator at 37˚C.
   NOTE: The overnight spent media should be aspirated and replaced with 100 µL of fresh, sterile RPMI-1640 media before use.

3. Preparation of cryptococcal cells

1. Streak out the test organism with an inoculation loop onto a sterile yeast-malt-extract (YM) agar plate.
2. Incubate the agar plate at 30˚C for 48 h.
3. Using an inoculation loop, inoculate a loopful of cells into 100 mL of yeast nitrogen base (YNB) broth supplemented with 4% glucose.
4. Grow the cells (pre-culture) overnight while shaking at 160 rpm at 30˚C.
5. The next day, inoculate 0.1 mL of the pre-culture into fresh YNB broth (main-culture) and allow cells to grow until the mid-exponential phase (under the same cultivation conditions).
6. Wash the cells twice with phosphate buffer solution (PBS).
7. Using a hemocytometer, standardize the cells to 1 x 10⁵ in PBS.

4. Staining of cryptococcal cells with the phagocytosis stain

1. Dispense 999 µL of the cells into a 1.5 mL plastic tube.
2. Add 1 µL of the pHrodo green zymosan A stain to the same tube.
3. Incubate the cells at room temperature for 1 h, in the dark, with slow agitation to react with the stain.
4. At the end of the incubation period, wash the cells thrice with PBS.
5. Dispense a 100 µL cell suspension to wells containing seeded macrophages.

5. Preparation of drugs

1. Prepare a stock aspirin solution using 1 mg of aspirin in 1000 mL of absolute ethanol to a final concentration of 1000 µg/mL.
2. Dilute the compound further in RPMI-1640 media to reach the desired drug concentrations.
   NOTE: The final amount of ethanol in RPMI-1640 media should never exceed 1%.Prepare a stock solution of ibuprofen using 1 mg of ibuprofen in 1000 mL of      dimethyl sulfoxide (DMSO).Dilute the compound further in RPMI-1640 media to reach the desired drug concentrations.
   NOTE: The final amount of DMSO in RPMI-1640 media should never exceed 1%. Prepare a 100 µL solution of the prepared drugs at quadruple the desired concentrations and dispense into appropriate wells containing seeded macrophages and cryptococcal cells.

6. The effect of chemo-sensitizing agents on macrophage phagocytosis

1. Incubate the plates with co-cultured cells in the presence of chemo-sensitizing agents, aspirin, or ibuprofen in a 5% CO₂ incubator at 37˚C for 2 h or 6 h.
2. At the end of the incubation period, measure the induced fluorescence (ex: 492 nm and em: 538 nm).
   NOTE: The pHrodo stain only fluoresces in acidic environments, such as inside the phagosome.