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

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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).

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