An Assay to Study the Impact of Photodynamic Treatment with a Photosensitizer on Macrophage Metabolic Activity
Published: February 29, 2024
Abstract
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.
Protocol
1. Safety considerations Personnel should use personal protective equipment (PPE). Handle the macrophages in a Class II Biological safety cabinet. Avoid self-exposure to UV light. 2. Preparation of macrophages Cultivate a murine macrophage cell line, RAW 264.7, on a tissue flask containing 10 mL of RPMI-1640 medium, supplemented with 20 mg/mL streptomycin, 2 mM L-glutamine, 20 U/mL penicillin and 10% fetal bov…
Representative Results
Figure 1 is a schematic representation that shows the macrophage metabolic activity protocol.
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
Tags
Cite This Article
An Assay to Study the Impact of Photodynamic Treatment with a Photosensitizer on Macrophage Metabolic Activity. J. Vis. Exp. (Pending Publication), e22204, doi: (2024).