Measuring Macrophage Phagocytic Efficiency with Primaquine and Photodynamic Therapy

Published: February 29, 2024

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 …

Offenlegungen

The authors have nothing to disclose.

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.

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Diesen Artikel zitieren
Measuring Macrophage Phagocytic Efficiency with Primaquine and Photodynamic Therapy. J. Vis. Exp. (Pending Publication), e22203, doi: (2024).

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