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

Published: February 29, 2024

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% …

Divulgaciones

The authors have nothing to disclose.

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. 

Tags

Play Video

Citar este artículo
The Effect of Aspirin and Ibuprofen on the Phagocytic Ability of Macrophages. J. Vis. Exp. (Pending Publication), e22205, doi: (2024).

View Video