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

Abstract

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

Materials

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.