effect of aspirin and ibuprofen on phagocytic ability of macrophages

Effect of Aspirin and Ibuprofen on Phagocytic Ability of Macrophages

Begin with a culture of the murine macrophage cell line, grown in complete RPMI 1640 medium, as described in the text protocol.
Additionally, prepare a culture of Cryptococcus neoformans resuspended in PBS to the desired concentration, as mentioned in the text protocol.&#160;
To stain the cryptococcal cells with the phagocytosis stain, dispense 999 microliters of the cells into a microcentrifuge tube. Add 1 microliter of the stain to the tube. Incubate the cells for 1 hour at room temperature, in the dark, with slow agitation.
After incubation, wash the cells by first centrifugation. Then, discard the supernatant. Resuspend the cells with PBS and repeat the washing step two additional times.
Dispense 100 microliters of the Cryptococcus neoformans cell suspension into the wells of the 96-well plate containing the cultured macrophages.
To study the effect of the test drugs on macrophage phagocytosis, dispense 100 microliters of the desired test drug into appropriate wells of the 96-well plate as described in the text protocol.
Incubate the plate in a carbon dioxide incubator for 2 to 6 hours. At the end of the incubation, measure the fluorescence from the phagocytic stain.

effect-of-aspirin-and-ibuprofen-on-phagocytic-ability-of-macrophages

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Immunodiagnostics

Cellular Immunodiagnostics

The Effect of Aspirin and Ibuprofen on the Phagocytic Ability of Macrophages

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1. Safety considerations
<ol>
	<li>Personnel should use personal protective equipment (PPE).</li>
	<li>Handle the pathogen and macrophages in a Class II Biological safety cabinet.</li>
</ol>
2. Preparation of macrophages
<ol>
	<li>Cultivate a murine macrophage cell line, RAW 264.7, in a tissue flask containing 10 mL of Roswell Park Memorial Institute-1640 (RPMI-1640) medium, supplemented with 20 mg/mL streptomycin, 2 mM L-glutamine, 20 U/mL penicillin and 10% fetal bovine serum (FBS).</li>
	<li>Place the flask in a 5% carbon dioxide (CO2) incubator set at 37&#730;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.&#160; 
	NOTE: A viability score that is above 85% is preferred. 
	Use a hemocytometer to adjust the cell concentration of macrophages to 1 &#215; 105 &#160;&#160;&#160; cells/mL using a 10 mL solution of fresh, sterile RPMI-1640 medium.&#160;Dispense 100 &#181;L suspension of cells into wells of a sterile 96-well flat-bottom microtiter plate. Incubate the plate overnight in a 5% CO2 incubator at 37&#730;C. 
	NOTE: The overnight spent media should be aspirated and replaced with 100 &#181;L of fresh, sterile RPMI-1640 media before use.</li>
</ol>
3. 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&#730;C for 48 h.</li>
	<li>Using an inoculation loop, inoculate a loopful of cells into 100 mL of yeast nitrogen base (YNB) broth supplemented with 4% glucose.</li>
	<li>Grow the cells (pre-culture) overnight while shaking at 160 rpm at 30&#730;C.</li>
	<li>The next day, inoculate 0.1 mL of the pre-culture into fresh YNB broth (main-culture) and allow cells to grow until the mid-exponential phase (under the same cultivation conditions).</li>
	<li>Wash the cells twice with phosphate buffer solution (PBS).</li>
	<li>Using a hemocytometer, standardize the cells to 1 x 105 in PBS.</li>
</ol>
4. Staining of cryptococcal cells with the phagocytosis stain
<ol>
	<li>Dispense 999 &#181;L of the cells into a 1.5 mL plastic tube.</li>
	<li>Add 1 &#181;L of the pHrodo green zymosan A stain to the same tube.</li>
	<li>Incubate the cells at room temperature for 1 h, in the dark, with slow agitation to react with the stain.</li>
	<li>At the end of the incubation period, wash the cells thrice with PBS.</li>
	<li>Dispense a 100 &#181;L cell suspension to wells containing seeded macrophages.</li>
</ol>
5. Preparation of drugs
<ol>
	<li>Prepare a stock aspirin solution using 1 mg of aspirin in 1000 mL of absolute ethanol to a final concentration of 1000 &#181;g/mL.</li>
	<li>Dilute the compound further in RPMI-1640 media to reach the desired drug concentrations. 
	NOTE: The final amount of ethanol in RPMI-1640 media should never exceed 1%.Prepare a stock solution of ibuprofen using 1 mg of ibuprofen in 1000 mL of &#160;&#160;&#160;&#160; dimethyl sulfoxide (DMSO).Dilute the compound further in RPMI-1640 media to reach the desired drug concentrations. 
	NOTE: The final amount of DMSO in RPMI-1640 media should never exceed 1%. Prepare a 100 &#181;L solution of the prepared drugs at quadruple the desired concentrations and dispense into appropriate wells containing seeded macrophages and cryptococcal cells.</li>
</ol>
6. The effect of chemo-sensitizing agents on macrophage phagocytosis
<ol>
	<li>Incubate the plates with co-cultured cells in the presence of chemo-sensitizing agents, aspirin, or ibuprofen in a 5% CO2 incubator at 37&#730;C for 2 h or 6 h.</li>
	<li>At the end of the incubation period, measure the induced fluorescence (ex: 492 nm and em: 538 nm). 
	NOTE: The pHrodo stain only fluoresces in acidic environments, such as inside the phagosome.</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>Yeast Nitrogen Base (YNB)</td>
			<td>Difco Laboratories</td>
			<td></td>
			<td>Used in the cultivation of cryptococcal cells&#160;</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>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>pHrodo green zymosan A stain</td>
			<td>Life Technologies</td>
			<td></td>
			<td>Used to stain internalised cells due to a change in pH&#160;&#160;</td>
		</tr>
		<tr>
			<td>Acetylsalicylic acid (Aspirin)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>The drug that is to be tested</td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Merk</td>
			<td></td>
			<td>Used to dissolve aspirin&#160;</td>
		</tr>
		<tr>
			<td>Ibuprofen</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Drug to be tested</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Merk&#160;</td>
			<td></td>
			<td>Used to dissolve ibuprofen</td>
		</tr>
		<tr>
			<td>96-well flat-bottom microtiter plates</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>15 mL centrifuge tube</td>
			<td>Lasec&#160;</td>
			<td></td>
			<td>Used for containing macrophages&#160;</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&#160;</td>
			<td></td>
			<td>Used to streak culture onto the agar plate</td>
		</tr>
		<tr>
			<td>Hemocytometer&#160;</td>
			<td>Marienfield&#160;</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>EZ Read 800 Research spectrophotometer&#160;</td>
			<td>Biochrom</td>
			<td></td>
			<td>To read the optical density&#160;</td>
		</tr>
		<tr>
			<td>Fluoroskan Ascent FL</td>
			<td>Thermo-Scientific</td>
			<td></td>
			<td>To measure fluorescence</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo-Fisher</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.&#160;</td>
		</tr>
	</tbody>
</table>

the effect of aspirin and ibuprofen on the phagocytic ability of macrophages

This video showcases a method to assess the impact of aspirin and ibuprofen, at their minimum inhibitory concentrations, on macrophage phagocytosis using pH-sensitive fluorescent dye-stained fungal cells. The drugs inhibit cyclooxygenase-2, decreasing prostaglandin formation and boosting macrophage phagocytosis, as observed through fluorescence measurements of pH-sensitive dye.

1. Safety considerations

	Personnel should use personal protective equipment (PPE).
	Handle the pathogen and macrophages in a Class II Biological safety ...

Begin with a fungal suspension of Cryptococcus. Add a solution of fungal-derived bioparticles conjugated with a pH-sensitive fluorescent dye to stain the Cryptococcus cells.
Wash to remove the unbound dye. Transfer the stained fungal suspension into a multi-well plate with adhered macrophages &#8212; or phagocytic immune cells.
Introduce the minimum inhibitory concentration of a drug, such as aspirin or ibuprofen, and incubate to facilitate the drug uptake.
The drug molecules interact with cyclooxygenase-2 &#8212; a prostaglandin-synthesizing enzyme, inhibiting its activity.
This prevents the conversion of arachidonic acid to prostaglandin, preserving intracellular cAMP levels and enhancing the macrophage phagocytic ability, facilitating the internalization of fungi.
The fungi-containing phagosome fuses with the lysosome &#8212; an acidic organelle, forming a phagolysosome with an acidic pH and allowing the dye to fluoresce.
Drug-treated cells exhibit a higher fluorescence than untreated cells, suggesting the drug&#8217;s efficacy in enhancing the phagocytic ability of macrophages.

the-effect-aspirin-ibuprofen-on-phagocytic-ability

129 vistas. Efecto de la aspirina y el ibuprofeno sobre la capacidad fagocítica de los macrófagos

Video: Efecto de la aspirina y el ibuprofeno sobre la capacidad fagocítica de los macrófagos - Experimento

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Evaluación de los efectos de drogas basados en anticuerpos en médula ósea murina y activación de los macrófagos peritoneales

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Investigación

JoVE Journal

Inmunología e Infección

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

Uso complementario de técnicas microscópicas y lectura de fluorescencia en el estudio de las interacciones Cryptococcus-Amoeba

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Fagocitosis del macrófago alveolar y remoción de bacterias en los ratones

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

The Effect of Aspirin and Ibuprofen on the Phagocytic Ability of Macrophages

Transcripción

Effect of Aspirin and Ibuprofen on Phagocytic Ability of Macrophages