effects of photodynamic treatment with primaquine on the phagocytic efficiency of murine macrophages

Effects of Photodynamic Treatment with Primaquine on the Phagocytic Efficiency of Murine Macrophages

Begin with a suspension of a murine macrophage cell line in complete RPMI 1640 medium as mentioned in the text protocol. Dispense 100 microliters of this cell suspension into the wells of a sterile 96-well flat-bottom culture plate. Incubate the plate overnight in an incubator.
The next day, remove the plate from the incubator. Replace the spent medium with 100 microliters of fresh RPMI-1640 medium. Then, dispense 100 microliters of cryptococcal cell suspension into the culture plate containing seeded macrophages.
To study the photodynamic effect, prepare the desired working concentrations of the photosensitizer. Add 100 microliters of working solution to the plate pre-seeded with macrophages and cryptococcal cells.
Incubate the plate in the dark for the cells to react with the drug. Post-incubation, expose the plates to UV light for two minutes.
After initiating photodynamic treatment, incubate the culture plates for 18 hours. Post-incubation, remove the supernatant, and wash the cells with 200 microliters of phosphate buffer solution.
Add 300 microliters of 0.1% Triton X-100 to the wells, and incubate at room temperature for 10 minutes.
Aspirate the contents of each well and dispense them into individual sterile 1.5-milliliter plastic tubes. Dilute the cells 10 times with distilled water, and pipette 50 microliters of the dilution onto individual yeast-malt-extract agar plates.
Use a spread plate method to create a confluent lawn of cells on the surface of the agar plate. Incubate the plates for 48 hours. Count the colony-forming units on the agar plates.

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Measuring Macrophage Phagocytic Efficiency with Primaquine and Photodynamic Therapy

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1. Preparation of Cryptococcal cells
<ol>
	<li>Streak out the test organism with an inoculation loop onto a sterile yeast-malt-extract (YM) agar plate.</li>
	<li>Incubate the agar plate at 30&#176;C for 48 h.</li>
	<li>Use an inoculation loop to scoop 5 single colonies and suspend them in 5 mL of sterile distilled water.</li>
	<li>Standardize the cells using a McFarland standard to obtain a cell concentration between 0.5 &#215; 105 and 2.5 &#215; 105 colony-forming units (CFU) per mL of RPMI-1640 medium.</li>
	<li>Dispense a 100 &#181;L cell suspension into wells of microtiter plates that contain seeded macrophages (see the section below).</li>
</ol>
2. Preparation of macrophages
<ol>
	<li>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.</li>
	<li>Place the flask in a 5% CO2 incubator set at 37&#176;C until 80% confluence is achieved.</li>
	<li>Gently scrape off the cells using a cell scraper.</li>
	<li>Transfer the suspended cells into a 15 mL centrifuge tube.</li>
	<li>Use the trypan blue stain to determine cell viability. 
	NOTE: A viability score that is above 85% is preferred.</li>
	<li>Use a hemocytometer to adjust the cell concentration of the macrophages to 1 &#215; 106 cells/mL using a 10 mL solution of fresh, sterile RPMI-1640 medium.</li>
	<li>Dispense 100 &#181;L suspension of cells into wells of a sterile 96-well flat-bottom microtiter plate.</li>
	<li>Incubate the plate overnight in a 5% CO2 incubator at 37&#176;C. 
	NOTE: Before use, the overnight spent media should be aspirated and replaced with 100 &#181;L of fresh, sterile Roswell Park Memorial Institute-1640 (RPMI-1640) media.</li>
</ol>
3. Compound preparation
<ol>
	<li>Dissolve test photosensitizers and primaquine (PQ) in distilled water to prepare a stock solution.</li>
	<li>Dilute the compound further in RPMI-1640 media. 
	NOTE: The final amount of distilled water in RPMI-1640 media should never exceed 1%.</li>
	<li>Dispense a 100 &#181;L solution of the prepared photosensitizer to quadruple the desired concentration into appropriate wells containing the already seeded macrophages and cryptococcal cells.</li>
</ol>
4. Implementation of photodynamic treatment (PDT)
<ol>
	<li>Set up the following experimental conditions:
	<ol>
		<li>Co-cultured cells with 0 &#181;M of PQ and exposed to dark light (DL) for 2 min (i.e., non-treated (non-Rx) cells)</li>
		<li>Co-cultured cells with 6 &#181;M and exposed to DL for 2 min (i.e., drug effect)</li>
		<li>Co-cultured cells 15 &#181;M of PQ and exposed to DL for 2 min (i.e., drug effect)</li>
		<li>Co-cultured cells with 30 &#181;M of PQ and exposed to DL for 2 min (i.e., drug effect)</li>
		<li>Co-cultured cells with 0 &#181;M of PQ and exposed to ultraviolet (UV) for 2 min (UV effect)</li>
		<li>Co-cultured cells with 6 &#181;M of PQ and exposed to ultraviolet light (UVL) for 2 min (i.e., PDT effect)</li>
		<li>Co-cultured cells with 15 &#181;M of PQ and exposed to UVL for 2 min (i.e., PDT effect)</li>
		<li>Co-cultured cells with 30 &#181;M 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 &#181;W/cm2.</li>
	</ol>
	</li>
</ol>
5. Counting colonies to determine the survival of Cryptococcal cells
<ol>
	<li>After initiating photodynamic treatment of the co-cultured cells as indicated above, incubate the plates at 37&#176;C in 5 % CO2 for 18 h.</li>
	<li>After 18 h, aspirate the supernatant.</li>
	<li>Wash the wells with 200 &#181;L of phosphate buffer solution (PBS) to wash off non-internalized cells. Repeat this step thrice.</li>
	<li>Add 300 &#181;L of 0.1% Triton X-100 to the wells and incubate at room temperature for 10 minutes.</li>
	<li>Aspirate the contents of the wells and dispense them into sterile 1.5 mL plastic tubes.</li>
	<li>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.</li>
	<li>Pipette 50 &#181;L of the dilution onto a Yeast Malt (YM) agar plate.</li>
	<li>Use a spread plate method to create a confluence lawn of cells on the surface of the agar plate.</li>
	<li>Incubate the plates at 30&#730;&#176;C for 48 h.</li>
	<li>Count the colony-forming units (CFUs) developed on the agar plates.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Merck</td>
			<td></td>
			<td>Used to prepare YM plates</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Merck</td>
			<td></td>
			<td>Used to prepare YM plates</td>
		</tr>
		<tr>
			<td>Malt extract</td>
			<td>Merck</td>
			<td></td>
			<td>Used to prepare YM plates</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Merck</td>
			<td></td>
			<td>Used to prepare YM plates</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td></td>
			<td>Used to prepare YM plates</td>
		</tr>
		<tr>
			<td>Trypan blue stain</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>To enumerate live and dead cells</td>
		</tr>
		<tr>
			<td>McFarland standard</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>To standardise cells</td>
		</tr>
		<tr>
			<td>Primaquine</td>
			<td>Merck</td>
			<td></td>
			<td>Used as a photosensitiser</td>
		</tr>
		<tr>
			<td>Phosphate buffer solution (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Wash and dilute cells</td>
		</tr>
		<tr>
			<td>RPMI- 1640 medium</td>
			<td>Biochrom</td>
			<td></td>
			<td>Cultivation media for macrophages</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Biochrom</td>
			<td></td>
			<td>Used to supplement the RPMI-1640 medium</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/L-glutamine</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Added to the RPMI-1640 medium</td>
		</tr>
		<tr>
			<td>0.1% Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Used to lyse macrophages</td>
		</tr>
		<tr>
			<td>96-well flat-bottom microtiter plate</td>
			<td>Greiner Bio-One</td>
			<td></td>
			<td>Used as a vessel within which reactions were carried-out</td>
		</tr>
		<tr>
			<td>Tissue flask</td>
			<td>Lasec</td>
			<td></td>
			<td>Used to cultivate macrophages</td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Lasec</td>
			<td></td>
			<td>Used to contain macrophages</td>
		</tr>
		<tr>
			<td>Cell scrapper</td>
			<td>Lasec</td>
			<td></td>
			<td>Used to detach macrophages from the tissue flask</td>
		</tr>
		<tr>
			<td>Inoculation loop</td>
			<td>Lasec</td>
			<td></td>
			<td>Used to streak culture onto the agar plate</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Marienfield</td>
			<td></td>
			<td>To determine the cell concentration of the macrophages</td>
		</tr>
		<tr>
			<td>1.5 mL plastic tubes</td>
			<td>Merck</td>
			<td></td>
			<td>Used as a vessel within which reactions were carried-out</td>
		</tr>
		<tr>
			<td>UVL</td>
			<td>ESCO</td>
			<td></td>
			<td>Used for the activation of the photosensitiser</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>ThermoFisher</td>
			<td></td>
			<td>Used to incubate the macrophages</td>
		</tr>
		<tr>
			<td>Class II biological safety cabinet</td>
			<td>ESCO</td>
			<td></td>
			<td>To safely work with the pathogens without causing harm to the user.</td>
		</tr>
	</tbody>
</table>

measuring macrophage phagocytic efficiency with primaquine and photodynamic therapy

The video demonstrates evaluating photodynamic treatment&#39;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.

1. Preparation of Cryptococcal cells

	Streak out the test organism with an inoculation loop onto a sterile yeast-malt-extract (YM) agar plate.
	Incubate ...

Begin with a multi-well plate containing adhered murine macrophages &#8212; or phagocytic immune cells.
Introduce a fungal cell suspension of Cryptococcus, interacting with macrophage receptors and internalizing via phagocytosis.
The phagosome fuses with the lysosome, forming a phagolysosome. The acidic environment of the phagolysosome favors fungal survival and multiplication.
Introduce a high concentration of primaquine &#8212; a photosensitizer, to the test wells, while the control lacks the photosensitizer. Incubate to facilitate primaquine uptake by macrophages.
Expose the plate to ultraviolet light for photodynamic treatment or PDT.
In the test well, primaquine forms radical cations that interact with molecular oxygen, producing reactive oxygen species, or ROS.
ROS induce fungal death, while anti-oxidative enzymes of macrophages neutralize ROS, preventing cellular damage.
Permeabilize the macrophages, releasing fungal cells.
Spread the diluted cell contents on selective agar plates, yielding distinct fungal colonies.
Fewer colonies on the test plate compared to the control indicate the enhanced macrophage phagocytic efficiency due to photodynamic treatment with primaquine.

measuring-macrophage-phagocytic-efficiency-with-primaquine

198 Views. Effects of Photodynamic Treatment with Primaquine on the Phagocytic Efficiency of Murine Macrophages

Video: Effects of Photodynamic Treatment with Primaquine on the Phagocytic Efficiency of Murine Macrophages - Experiment

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

JoVE Journal

Cancer Research

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

Biology

Assessing the Putative Anticryptococcal Properties of Crude and Clarified Extracts from Mollusks

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The widespread use of antifungals in clinical settings, their use in agriculture, and strain hybridization have led to increased evolution of resistance. This rising rate of resistance against antifungals is a growing concern among clinicians and scientists worldwide, and there is heightened urgency to develop novel antifungal therapies. For instance, C. neoformans produces several virulence factors, including intra- and extra-cellular enzymes (e.g., peptidases) with roles in tissue degradation, cellular regulation, and nutrient acquisition. The disruption of such peptidase activity by inhibitors perturbs fungal growth and proliferation, suggesting this may be an important strategy for combating the pathogen. Importantly, invertebrates such as mollusks produce peptidase inhibitors with biomedical applications and anti-microbial activity, but they are underexplored in terms of their usage against fungal pathogens. In this protocol, a global extraction from mollusks was performed to isolate potential peptidase inhibitors in crude and clarified extracts, and their effects against classical cryptococcal virulence factors were assessed. This method supports the prioritization of mollusks with antifungal properties and provides opportunities for the discovery of anti-virulence agents by harnessing the natural inhibitors found in mollusks.

The human fungal pathogen Cryptococcus neoformans produces a variety of virulence factors (e.g., peptidases) to promote its survival within the host. Environmental niches represent a promising source of novel natural peptidase inhibitors. This protocol outlines the preparation of extracts from mollusks and the assessment of their effect on fungal virulence factor production.

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The ...

Measuring Macrophage Phagocytic Efficiency with Primaquine and Photodynamic Therapy

Transcript

Effects of Photodynamic Treatment with Primaquine on the Phagocytic Efficiency of Murine Macrophages