Name: visualizing the early stages of phagocytosis
Uploaded: 2017-02-03T12:30:00
Description: Watch this Scientific Journal Video about Visualizing the Early Stages of Phagocytosis at JoVE.com

We thank Wendy Salmon and Nicki Watson of the Keck facility at the Whitehead Institute of MIT for imaging.

1. Preparation of DC2.4 and RAW 264.7 Cell Lines
NOTE: The macrophage-like cell line RAW 264.7 and the dendritic cell line DC2.4 are both murine origin, and the following conditions were used to grow the cells.
<ol>
	<li>Grow RAW 264.7 cells in DMEM (Dulbecco&#39;s Minimal Eagle&#39;s medium) supplemented with 10% inactivated Fetal Bovine Serum (FBS) at 37 &#176;C in a humidified incubator with 5% CO2.</li>
	<li>Grow DC2.4 cells in similar conditions with that of RAW 264.7 cells, ...

<ol>
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Professional phagocytes, such as macrophages and dendritic cells, engulf and eliminate invading pathogens therefore making phagocytosis an important component of the host defense system. During this process phagocytes undergo extensive membrane reorganization and cytoskeleton rearrangement at their cell surface8,19,20. To better understand this dynamic process, visualization of the different stages of phagocytosis is essential. ...

Despite the continuous exposure to pathogens such as bacteria, viruses and fungi, our body is well equipped with immune mechanism that provide protection against infection. The innate immune system is the first line of defense against invading pathogens and relies mainly on phagocytic cells that recognize and internalize foreign targets.
Phagocytosis is an evolutionarily conserved cellular process that encompasses the engulfment of unwanted particulates greater than 0.5 &#181;m. Phagocytic cells express a wide range of immune receptors (also known as pattern recognition receptors, PRRs) at the cell surface that enable them to recognize pathogen-associated molecular patterns (PAMPs) present on pathogens prior to engulfment3. Pathogen binding is followed by receptor clustering at the cell surface and triggers the formation of a phagocytic cup. This results in actin-driven membrane remodeling that protrudes around the target, eventually enveloping it and pinching-off to form a discrete phagosomal vacuole2,5. The phagosome then matures and acidifies by subsequent fusion with late endosomes and lysosomes that forms phagolysosome6.
Although phagocytosis is described as receptor-mediated and actin-driven event, this process also relies on spatial-temporal modification of lipids that compose the plasma membrane, such as phosphoinositides (PIs) and sphingolipids7,8. While actin polymerization is dictated by a local accumulation of phosphoinositol-4,5-biphosphate (PI(4,5)P2) at the base of the phagocytic cup, actin depolymerization depends on the conversion of (PI(4,5)P2 to phosphoinositol-3,4,5-biphosphate (PI(3,4,5)P3)3,9. Both modifications are essential as the former leads to successful extension of pseudopods around the target and the latter enables sinking of particles in the cytosol of the phagocyte10.
Cells that have the ability to phagocytose are either professional phagocytes, like macrophages/monocytes, granulocytes/neutrophils, and dendritic cells (DCs) or nonprofessional phagocytes, such as fibroblast and epithelial cells11. Phagocytosis performed by all phagocytes plays a central role in tissue maintenance and remodeling, while phagocytosis performed by professional phagocytes is responsible for the coordination of the innate and adaptive immune response against pathogens. Professional phagocytes do not only engulf and kill the pathogen, but also present antigens to the lymphoid cells of the adaptive immune system. This contributes to the release of pro-inflammatory cytokines and to the engagement of lymphoid cells, therefore leading to the successful blockade of infection12.
Conventional biochemical techniques have been instrumental in gaining knowledge regarding the molecular mechanism of different cellular processes during phagocytosis, such as post-translational modifications and various high-affinity associations between proteins. However, it is difficult to obtain information regarding the spatial and temporal dynamics of phagocytic events using the conventional biochemical methods. Live cell imaging not only allows us to monitor cellular events in a time sensitive manner but also enables us to gain information at a single cell level. Here we describe a method to investigate the different stages of phagocytosis, as well as to analyze the whole process spatiotemporally using confocal microscopy.

<table border="0" cellpadding="0" cellspacing="0" width="988">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">&#946;-Mercaptoethanol</td>
			<td style="width:253px;">AppliChem</td>
			<td style="width:332px;">A1108</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Bovine serum albumin (BSA)</td>
			<td>Cell Signaling Technology&#160;</td>
			<td>9998</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dilactate)</td>
			<td>ThermoFisher</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dimethyl sulfoxide (DMSO)</td>
			<td>ThermoFisher</td>
			<td>BP-231-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">DMEM (Dulbecco&#8217;s Minimal Eagle&#8217;s medium)</td>
			<td style="width:253px;">Gibco</td>
			<td style="width:332px;">11965</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">PBS, 1X (Phosphate- Buffered Saline)</td>
			<td>Corning cellgro</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal Bovine serum</td>
			<td>Sigma Aldrich</td>
			<td>12003C</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">FITC-coupled IgG-coated latex beads</td>
			<td style="width:253px;">Cayman</td>
			<td style="width:332px;">500290</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">L-Glutamine 200mM (100X)</td>
			<td>ThermoFisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Paraformaldehyde Solution (4% in PBS)</td>
			<td>Affymetrix</td>
			<td>19943 1 LT</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Penicillin-streptomycin (10&#39;000U/mL)</td>
			<td>ThermoFisher</td>
			<td style="width:332px;"><a href="https://www.fishersci.com/shop/products/gibco-penicillin-streptomycin-10-000-u-ml-3/15140122">15-140-122</a></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Phalloidin-Alexa Fluor 488</td>
			<td style="width:253px;">ThermoFisher</td>
			<td style="width:332px;">A12379</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">RAW 264.7 cells</td>
			<td style="width:253px;">ATCC</td>
			<td style="width:332px;">TIB-71</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">RPMI (Roswell Park Memorial Institute)</td>
			<td style="width:253px;">Gibco</td>
			<td style="width:332px;">61870</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Saponin</td>
			<td>Sigma Aldrich</td>
			<td>S7900</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Trypan blue solution (0.4% (w/v) in PBS)</td>
			<td>Corning cellgro</td>
			<td>MT25900CI</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Trypsin-EDTA (1X) (0.05%)</td>
			<td>ThermoFisher</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Tween 20 Surfact-Amps Detergent Solution</td>
			<td>ThermoFisher</td>
			<td>&#160;85114</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Zymosan-Alexa Fluor 594</td>
			<td style="width:253px;">ThermoFisher</td>
			<td style="width:332px;">Z23374</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:315px;">Chambered 1.0 Borosilicate Coverglass system (8 chambers)</td>
			<td>ThermoFisher</td>
			<td>155361</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glasstic slide 10 with grids</td>
			<td>Hycor</td>
			<td>87144</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing the early stages of phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Microscope-based method to monitor the different stages of phagocytosis is presented. The different events during the phagocytosis of various fluorescent particulates by DC2.4 cells are shown. Using the techniques described here, we investigated the role of sphingolipids in the early stages of phagocytosis. For this purpose, DC2.4 dendritic cells genetically deficient in Sptlc2, the enzyme that catalyzes the first and rate-limiting step in the sphingolipid biosynthetic pathway, were used. As compared to wild type cells, ...

Watch this Scientific Journal Video about Visualizing the Early Stages of Phagocytosis at JoVE.com

Visualizing the Early Stages of Phagocytosis

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

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