An Assay to Study the Impact of Photodynamic Treatment with a Photosensitizer on Macrophage Metabolic Activity

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.

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