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

Overview

This video showcases a method to assess the impact of aspirin and ibuprofen, at their minimum inhibitory concentrations, on macrophage phagocytosis using pH-sensitive fluorescent dye-stained fungal cells. The drugs inhibit cyclooxygenase-2, decreasing prostaglandin formation and boosting macrophage phagocytosis, as observed through fluorescence measurements of pH-sensitive dye.

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
Agar	Merck		Used to prepare YM plates
Yeast extract	Merck		Used to prepare YM plates
Malt extract	Merck		Used to prepare YM plates
Peptone	Merck		Used to prepare YM plates
Yeast Nitrogen Base (YNB)	Difco Laboratories		Used in the cultivation of cryptococcal cells
Glucose	Merck		Used to prepare YM plates
Trypan blue stain	Sigma-Aldrich		To enumerate live and dead cells
Phosphate buffer solution (PBS)	Sigma-Aldrich		Wash and dilute cells
RPMI- 1640 medium	Biochrom		Cultivation media for macrophages
Fetal bovine serum (FBS)	Biochrom		Used to supplement the RPMI-1640 medium
Penicillin/Streptomycin/L-glutamine	Sigma-Aldrich		Added to the RPMI-1640 medium
pHrodo green zymosan A stain	Life Technologies		Used to stain internalised cells due to a change in pH
Acetylsalicylic acid (Aspirin)	Sigma-Aldrich		The drug that is to be tested
Absolute ethanol	Merk		Used to dissolve aspirin
Ibuprofen	Sigma-Aldrich		Drug to be tested
Dimethyl sulfoxide (DMSO)	Merk		Used to dissolve ibuprofen
96-well flat-bottom microtiter plates	Greiner Bio-One		Used as a vessel within which reactions were carried-out
Tissue flask	Lasec		Used to cultivate macrophages
15 mL centrifuge tube	Lasec		Used for containing macrophages
Cell scrapper	Lasec		Used to detach macrophages from the tissue flask
Inoculation loop	Lasec		Used to streak culture onto the agar plate
Hemocytometer	Marienfield		To determine the cell concentration of the macrophages
1.5 mL plastic tubes	Merck		Used as a vessel within which reactions were carried-out
EZ Read 800 Research spectrophotometer	Biochrom		To read the optical density
Fluoroskan Ascent FL	Thermo-Scientific		To measure fluorescence
CO2 incubator	Thermo-Fisher		Used to incubate the macrophages
Class II biological safety cabinet	ESCO		To safely work with the pathogens without causing harm to the user.