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 …
Divulgaciones
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.