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.
1. Safety considerations
2. Preparation of macrophages
3. Preparation of cryptococcal cells
4. Staining of cryptococcal cells with the phagocytosis stain
5. Preparation of drugs
6. The effect of chemo-sensitizing agents on macrophage phagocytosis
The authors have nothing to disclose.
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. |