The video demonstrates a technique for evaluating the influence of photodynamic treatment (PDT) with a photosensitizer on the metabolic activity of murine macrophages. The cells undergo treatment with the photosensitizer followed by exposure to UV light, initiating PDT. This process triggers the photosensitizer to generate reactive oxygen species (ROS) within the cells, causing cellular toxicity. The impact of ROS is then assessed by enzymatic antioxidants through a colorimetric assay, providing insights into the metabolic activity within the cells.
1. Safety considerations
2. Preparation of macrophages
3. Compound preparation
4. Implementation of PDT
5. Macrophage metabolic activity assay
Table 1. An illustration of the experimental conditions to be set up before initiating the experiment. In this protocol, PQ was tested at 0, 6, 60 and 600 µM.
Macrophages seeded in the UVL-only plate | Macrophages seeded in the PQ-only plate | Macrophages seeded in the PDT-only plate |
Macrophages with 0 µM of PQ | Macrophages with 0 µM of PQ | – |
– | Macrophages with 6 µM of PQ | Macrophages with 6 µM of PQ |
– | Macrophages with 60 µM of PQ | Macrophages with 60 µM of PQ |
– | Macrophages with 600 µM of PQ | Macrophages with 600 µM of PQ |
NOTE: All the seeded plates should be allowed to stand for 30 min under ambient light and temperature. During this period, cells in the 1) UVL-only plate will react to ambient light, 2) PQ-only plate will react to different PQ concentrations, and 3) PDT-only plate will, likewise, react to different PQ concentrations under ambient light.
Figure 1 is a schematic representation that shows the macrophage metabolic activity protocol.
The authors have nothing to disclose.
Primaquine | Merck | Used as a photosensitizer | |
Phosphate buffer solution (PBS) | Sigma-Aldrich | Wash and dilute cells | |
2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) | Invitrogen | Used to measure the metabolic activity of the macrophages | |
Menadione | Sigma-Aldrich | Used in conjunction with XTT | |
50 mL centrifuge tube | Lasec | Used to contain macrophages | |
Haemocytometer | Marienfield | To determine the cell concentration of the macrophages | |
RPMI- 1640 medium | Biochrom | Cultivation media for macrophages | |
Fetal bovine serum (FBS) | Biochrom | Used to supplement the RPMI-1640 medium | |
5% CO2 incubator | Thermo-Fisher | Used to incubate the macrophages | |
Cell scrapper | Lasec | Used to detach macrophages from the tissue flask | |
Trypan blue stain | Sigma-Aldrich | To enumerate live and dead cells | |
Penicillin/Streptomycin/ L-glutamine | Sigma-Aldrich | Added to the RPMI-1640 medium | |
96-well flat-bottom microtiter plate | Greiner Bio-One | Used as a vessel within which reactions were carried-out | |
1.5 mL plastic tubes | Merck | Used as a vessel within which reactions were carried-out | |
UVL | ESCO | Used as a light source to photosensitise PQ | |
EZ Read 800 spectrophotometer | Biochrom | To read the optical density | |
Fluoroskan Ascent FL microplate reader | Thermo Scientific | To measure fluorescence |