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