Measuring Macrophage Phagocytic Efficiency with Primaquine and Photodynamic Therapy

Abstract

The video demonstrates evaluating photodynamic treatment's impact on murine macrophage phagocytic efficiency with primaquine. Treated, infected macrophages generate reactive oxygen species, leading to selective fungal death, evident by reduced colonies on the plate.

Protocol

1. Preparation of Cryptococcal cells Streak out the test organism with an inoculation loop onto a sterile yeast-malt-extract (YM) agar plate. Incubate the agar plate at 30°C for 48 h. Use an inoculation loop to scoop 5 single colonies and suspend them in 5 mL of sterile distilled water. Standardize the cells using a McFarland standard to obtain a cell concentration between 0.5 × 105 and 2.5 × 105 colony-forming units …

Materials

Agar	Merck		Used to prepare YM plates
Yeast extract	Merck		Used to prepare YM plates
Malt extract	Merck		Used to prepare YM plates
Peptone	Merck		Used to prepare YM plates
Glucose	Merck		Used to prepare YM plates
Trypan blue stain	Sigma-Aldrich		To enumerate live and dead cells
McFarland standard	Fisher Scientific		To standardise cells
Primaquine	Merck		Used as a photosensitiser
Phosphate buffer solution (PBS)	Sigma-Aldrich		Wash and dilute cells
RPMI- 1640 medium	Biochrom		Cultivation media for macrophages
Fetal bovine serum (FBS)	Biochrom		Used to supplement the RPMI-1640 medium
Penicillin/Streptomycin/L-glutamine	Sigma-Aldrich		Added to the RPMI-1640 medium
0.1% Triton X-100	Sigma-Aldrich		Used to lyse macrophages
96-well flat-bottom microtiter plate	Greiner Bio-One		Used as a vessel within which reactions were carried-out
Tissue flask	Lasec		Used to cultivate macrophages
50 mL centrifuge tube	Lasec		Used to contain macrophages
Cell scrapper	Lasec		Used to detach macrophages from the tissue flask
Inoculation loop	Lasec		Used to streak culture onto the agar plate
Hemocytometer	Marienfield		To determine the cell concentration of the macrophages
1.5 mL plastic tubes	Merck		Used as a vessel within which reactions were carried-out
UVL	ESCO		Used for the activation of the photosensitiser
CO2 incubator	ThermoFisher		Used to incubate the macrophages
Class II biological safety cabinet	ESCO		To safely work with the pathogens without causing harm to the user.