Measuring Macrophage Phagocytic Efficiency with Primaquine and Photodynamic Therapy

Overview

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

1. Streak out the test organism with an inoculation loop onto a sterile yeast-malt-extract (YM) agar plate.
2. Incubate the agar plate at 30°C for 48 h.
3. Use an inoculation loop to scoop 5 single colonies and suspend them in 5 mL of sterile distilled water.
4. Standardize the cells using a McFarland standard to obtain a cell concentration between 0.5 × 10⁵ and 2.5 × 10⁵ colony-forming units (CFU) per mL of RPMI-1640 medium.
5. Dispense a 100 µL cell suspension into wells of microtiter plates that contain seeded macrophages (see the section below).

2. Preparation of macrophages

1. 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 bovine serum.
2. Place the flask in a 5% CO₂ incubator set at 37°C until 80% confluence is achieved.
3. Gently scrape off the cells using a cell scraper.
4. Transfer the suspended cells into a 15 mL centrifuge tube.
5. Use the trypan blue stain to determine cell viability.
   NOTE: A viability score that is above 85% is preferred.
6. Use a hemocytometer to adjust the cell concentration of the macrophages to 1 × 10⁶ cells/mL using a 10 mL solution of fresh, sterile RPMI-1640 medium.
7. Dispense 100 µL suspension of cells into wells of a sterile 96-well flat-bottom microtiter plate.
8. Incubate the plate overnight in a 5% CO₂ incubator at 37°C.
   NOTE: Before use, the overnight spent media should be aspirated and replaced with 100 µL of fresh, sterile Roswell Park Memorial Institute-1640 (RPMI-1640) media.

3. Compound preparation

1. Dissolve test photosensitizers and primaquine (PQ) in distilled water to prepare a stock solution.
2. Dilute the compound further in RPMI-1640 media.
   NOTE: The final amount of distilled water in RPMI-1640 media should never exceed 1%.
3. Dispense a 100 µL solution of the prepared photosensitizer to quadruple the desired concentration into appropriate wells containing the already seeded macrophages and cryptococcal cells.

4. Implementation of photodynamic treatment (PDT)

1. Set up the following experimental conditions:
   1. Co-cultured cells with 0 µM of PQ and exposed to dark light (DL) for 2 min (i.e., non-treated (non-Rx) cells)
   2. Co-cultured cells with 6 µM and exposed to DL for 2 min (i.e., drug effect)
   3. Co-cultured cells 15 µM of PQ and exposed to DL for 2 min (i.e., drug effect)
   4. Co-cultured cells with 30 µM of PQ and exposed to DL for 2 min (i.e., drug effect)
   5. Co-cultured cells with 0 µM of PQ and exposed to ultraviolet (UV) for 2 min (UV effect)
   6. Co-cultured cells with 6 µM of PQ and exposed to ultraviolet light (UVL) for 2 min (i.e., PDT effect)
   7. Co-cultured cells with 15 µM of PQ and exposed to UVL for 2 min (i.e., PDT effect)
   8. Co-cultured cells with 30 µM of PQ and exposed to UVL for 2 min (i.e., PDT effect)
      NOTE: The co-cultured cells must be allowed to react with PQ for 30 min in the dark (where applicable), before exposure to UVL (where applicable).
      NOTE: In the current work, we determined that PQ has a UV/Vis absorption of 260 nm, which is in the UV range of the germicidal lamp. Due to the latter, we used a germicidal ultraviolet C (UVC) lamp fitted in a Class II Biological safety cabinet as a light source to implement PDT. The lamp is reported to have a nominal power of 30 watts. The cells were kept at approximately 20 cm from the lamp in the current study. At this distance, the lamp is estimated to have a radiation output of 625 µW/cm².

5. Counting colonies to determine the survival of Cryptococcal cells

1. After initiating photodynamic treatment of the co-cultured cells as indicated above, incubate the plates at 37°C in 5 % CO₂ for 18 h.
2. After 18 h, aspirate the supernatant.
3. Wash the wells with 200 µL of phosphate buffer solution (PBS) to wash off non-internalized cells. Repeat this step thrice.
4. Add 300 µL of 0.1% Triton X-100 to the wells and incubate at room temperature for 10 minutes.
5. Aspirate the contents of the wells and dispense them into sterile 1.5 mL plastic tubes.
6. Prepare a 1 in 10 dilution of the cells with distilled water.
   NOTE: Adjust the dilution so that it is possible to recover between 30 and 300 colonies on agar plates.
7. Pipette 50 µL of the dilution onto a Yeast Malt (YM) agar plate.
8. Use a spread plate method to create a confluence lawn of cells on the surface of the agar plate.
9. Incubate the plates at 30˚°C for 48 h.
10. Count the colony-forming units (CFUs) developed on the agar plates.

Materials

Name	Company	Catalog Number	Comments
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.