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 …
Declarações
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. |
Tags
Citar este artigo
Measuring Macrophage Phagocytic Efficiency with Primaquine and Photodynamic Therapy. J. Vis. Exp. (Pending Publication), e22203, doi: (2024).