An Assay to Study the Impact of Photodynamic Treatment with a Photosensitizer on Macrophage Metabolic Activity
Transcript
Begin with a culture of the murine macrophage cell line, grown in complete RPMI 1640 medium, in a sterile 96-well flat-bottom microtiter plate, as described in the text protocol.
Additionally, prepare a stock solution of the photosensitizer by dissolving the powder in distilled water. Prepare serial dilutions of the photosensitizer solution using RPMI 1640 medium.
Now, add 100 microliters of the photosensitizer solution to appropriate wells of the microtiter plate seeded with macrophages. Incubate the culture under ambient light and temperature.
To test the effect of UV light, expose the photosensitizer-treated macrophage culture to the UV light.
To assess the metabolic activity of the treated macrophages, add 50 microliters of XTT reagent to all the wells containing the irradiated cells.
Add four microliters of menadione to improve the reduction of the XTT salt. Incubate the plates for 3 hours at 37 degrees Celsius with 5 percent carbon dioxide.
After incubation, measure the product formation by reading the absorbance at 492 nanometers.
