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, except use RPMI (Roswell Park Memorial Institute) medium supplemented with L-Glutamine instead of DMEM.</li>
</ol>
2. Preparation of Fluorescent Conjugated Particulates: C. albicans, Zymosan, IgG-coated Beads
<ol>
	<li>Growing Candida albicans
	<ol>
		<li>Inoculate C. albicans strain SC5314 from a frozen glycerol stock in 25 ml of YPD supplemented with Uri (2% Bacto-peptone, 1% yeast extract, 2% glucose and 80 &#181;g/ml Uridine) overnight at 30 &#176;C. 
		NOTE: Avoid growing the Candida culture more than an OD600 of 12.</li>
		<li>Take 1 ml of the C. albicans and spin it down at 2,000 x g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant and re-suspend the pellet with 1 ml of PBS.</li>
		<li>Collect the pellet by centrifugation at 2,000 x g for 5 min at room temperature.</li>
		<li>Repeat step 2.1.4.</li>
		<li>Calculate the multiplicity of infection (MOI) of C. albicans in relation to the number of phagocytes and proceed with the phagocytosis assay as described in section 3. An OD600 of 1 is equivalent to 2.5 x 107 Candida albicans per 1 ml. 
		NOTE: Generation of Candida-BFP is described in reference13.</li>
	</ol>
	</li>
	<li>Preparation of Zymosan and IgG-coated Beads 
	NOTE: Zymosan is a &#946;-glucan-containing fungal carbohydrate particulate preparation that derived from Saccharomyces cerevisiae. Zymosan is known to evoke inflammatory signals in macrophages and dendritic cells, and it has been used extensively in phagocytosis studies 13,14,15.
	<ol>
		<li>Briefly vortex the tube that contains the particulate, and aliquot an amount sufficient for an experiment into a 1.5 ml tube. 
		NOTE: Typically we use 1:10 ratio (for one cell, add 10 zymosan particles or IgG-coated beads).</li>
		<li>Add 1 ml of ice cold PBS, and collect the particles by centrifuging for 1 min at 10,000 x g.</li>
		<li>Aspirate the supernatant and resuspend the pellet in 1 ml ice-cold PBS.</li>
		<li>Repeat steps 2.2.2-2.2.3 
		NOTE: Proceed to step 2.2.5 or store the tube at 4 &#176;C until use.</li>
		<li>Sonicate the particulates for 10 sec in ultrasonic bath sonicator (120 V, 50-60 kHz) in order to avoid any particle aggregation.</li>
	</ol>
	</li>
</ol>
3. Phagocytosis
NOTE: Phagocytosis is a complex process that begins with binding of the particles on the cell surface of the phagocytes through interaction of PRRs with ligands on the surface of the particle. Binding is followed by the assembly of actin and its associated proteins at the site of contact, and the formation of a phagocytic cup. The subsequent actin disassembly occurs at the phagosome and results in the complete engulfment of the particulate. Below we describe the different stages of phagocytosis.
<ol>
	<li>Binding Assay 
	NOTE: The purpose of this experiment is to monitor the first step of phagocytosis that involves the interaction between the ligands on the target particles and the receptors on the phagocytes.
	<ol>
		<li>Seed about 5 x 104 cells in a chamber slide so that they are 60-70% confluent at the time of the experiment. Alternatively, plate cells on cover slips (in 24- or 12-well format) or glass-bottom 96-well plates. Incubate cells overnight at 37 &#176;C humidified incubator with 5% CO2. 
		NOTE: Cells can be counted using hemocytometer or a similar technique.</li>
		<li>Place the chamber slide (or plate) at 10 &#176;C for 10 min.</li>
		<li>Gently remove the medium from each well using a pipette or aspirator. 
		CAUTION: Perform this step carefully as each well of a chambered coverglass system is small; it is easy to perturb cells with the tip of a pipette or a strong airflow of an aspirator.</li>
		<li>Mix the fluorescently labeled particulates with a cell culture medium (prepared as described above). Add Candida-BFP (at an MOI of 10), fluorescently conjugated-zymosan or latex beads-rabbit IgG-FITC (10 particles per cell) to the cells. Use a final volume of 200 &#181;l media for one chamber well to ensure all cells are covered.</li>
		<li>Spin down the chamber slide at 277 x g for 3 min at 10 &#176;C. 
		NOTE: This centrifugation step helps to increase the contact between the particulates and the cells, and it synchronizes the binding process.</li>
		<li>Immediately transfer the chamber slide to a 37 &#176;C humidified incubator with 5% CO2 for 5 min.</li>
		<li>Remove the chamber slide from the incubator and aspirate the media.</li>
		<li>Wash the cells 3x with ice-cold PBS. 
		NOTE: Check the presence of unbound particles with a light microscope and wash more times with PBS if necessary. It is essential to remove unbound particles from the well to minimize background noise during the analysis.</li>
		<li>Fix cells by adding 500 &#181;l 4% paraformaldehyde (PFA) diluted in PBS by incubating for 30 min at room temperature. 
		CAUTION: This step requires the use of 4% PFA which is highly toxic, corrosive, and potential carcinogen. Proper caution should be taken when using this liquid. Also, protect samples from light.</li>
		<li>Wash the cells by adding 500 &#181;l of PBS into the chamber and aspirate the solution gently.</li>
		<li>Repeat 3.1.9 step twice. 
		NOTE: At this step, refill each vial immediately after removing the previous PBS.</li>
		<li>Stop the fixation by incubating with 500 &#181;l of 50 mM NH4Cl in PBS for 10 min.</li>
		<li>Wash cells twice with PBS as described in step 3.1.10.</li>
		<li>Permeabilize cells by adding 250 &#181;l binding buffer (0.1% saponin, 0.2% BSA in PBS) for 30 min at room temperature.</li>
		<li>Stain F-actin by adding fluorescently conjugated phalloidin (diluted 1:500 in binding buffer). Incubate cells for 1 hr at room temperature.</li>
		<li>Wash cells three times with PBS as described in step 3.1.10.</li>
		<li>Capture images using a confocal microscope (63X objective, and 402 nm and 488 nm laser) and analyze images using ImageJ16. 
		NOTE: A successful particle binding is characterized by the presence of a phagocytic cup at the site where the particulate contact the phagocytic cell (Figure 1). See step 3.2.5.</li>
	</ol>
	</li>
	<li>Phagocytosis Uptake Assay 
	NOTE: This experiment is designed to monitor the complete internalization of fluorescently labeled particulates by phagocytes using confocal microscopy (Figure 2).
	<ol>
		<li>Seed about 5 x 104 cells in a chamber slide so that they are 60-70% confluent at the time of the experiment. Alternatively, plate cells on cover slips (in 24- or 12-well format) or glass-bottom 96-well plates. Incubate cells overnight at 37 &#176;C humidified incubator with 5% CO2. 
		NOTE: Cells can be counted using hemocytometer or a similar technique.</li>
		<li>Keep the chamber slides (or plate) at 10 &#176;C for 10 min. 
		NOTE: Cooling is necessary in order to block endocytosis as well as unwanted phagocytosis, and subsequently to have a synchronized phagocytosis.</li>
		<li>Perform steps from 3.1.3 to 3.1.5</li>
		<li>Transfer the chamber slide to a 37 &#176;C humidified incubator with 5% CO2, and incubate for different time points. Fix cells after 30, 60 and 90 min post infection. Observe C. albicans begin to form hyphae at 60 min post infection; phagocytes begin to show cell death (pyroptosis) after 90 min8. 
		NOTE: This is a recommendation, and optimization is essential depending on the cell type and the particulate used.</li>
		<li>Perform steps from 3.1.7 to 3.1.15. Analyze images using ImageJ16. 
		NOTE: a successful uptake is characterized by a complete internalization of a particulate inside the cytoplasm of a phagocyte (Figure 2). Staining the phagocytes with cell surface marker such as phalloidin helps to delineate the contour of cells. It is also essential to perform z-stacks in order to determine whether the particulate is bound to cell surface or completely internalized.</li>
	</ol>
	</li>
	<li>Live Imaging of Phagocytosis 
	NOTE: In this protocol we describe the use of confocal microscopy to capture the different stages of phagocytosis using fluorescently conjugated zymosan. We use the dendritic cell line DC2.4 that stably expresses mCherry-tagged F-actin8, a biosensor that shows the distribution of filamentous actin in living cells. Since phagocytosis is an actin-driven process the live imaging in this cell not only enables us to capture the movement and dynamics of F-actin network, but also to visualize the different phagocytic events within the living cells (Figure 3, Movie 1).
	<ol>
		<li>Seed about 5 x 104 cells in a chamber slide at a concentration so that they are 60-70% confluent at the time of the experiment. Incubate cells overnight at 37 &#176;C humidified incubator with 5% CO2. 
		NOTE: Cells can be counted using hemocytometer or a similar technique.</li>
		<li>Keep the chamber slides on ice for 10 min. 
		NOTE: Cooling is necessary in order to block unwanted uptake, and to have a synchronized phagocytosis.</li>
		<li>Perform steps from 3.1.3 to 3.1.5</li>
		<li>Pre-warm the microscope stage to 37 &#176;C and equilibrate the chamber with 5% CO2. Wait 30-45 min for the temperature and the CO2 to stabilize before starting the imaging. 
		NOTE: Since the CO2 and temperature levels are critical for phagocytosis, it is important to turn on the microscope heater prior to the experiment to equilibrate the environmental chamber.</li>
		<li>Place the chamber slide containing the cells in the microscope incubator enclosure equilibrated at 37 &#176;C and 5% CO2.</li>
		<li>Perform live imaging17,18. 
		NOTE: The settings for the laser power (gain, pinhole, etc.) can vary depending on the type of the microscope and fluorescent probe used. Therefore, we recommend consulting the manual of the microscope for optimal condition for your live imaging 17,18.
		<ol>
			<li>Use confocal laser scanning microscope (and the associated software) for live imaging. Use 63X 1.4NA oil objective with a pinhole of 1-1.1 Airy units.</li>
			<li>To start the imaging, used the bright field to find a representative region on the chamber slide. Next, select the field by clicking the Position option on the software. To detect GFP in Track 1 used the filter set for 488 nm laser with a beam splitter at 582 nm, to detect wavelengths between 488 and 582 nm.</li>
			<li>In Track 2 select a filter set optimal for mCherry (RFP) using a beam splitter at 578 nm and detecting wavelengths between 578 and 600 nm. Use 488 nm and 578 nm lasers with 50% laser power and a gain output of 600-700. Obtain images every 30 sec for a total of 90 min.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+phagocytosis+in+macrophages.">Mechanisms of phagocytosis in macrophages.</a> Annu Rev. Immunol. 17, 593-623 (1999).</li><li>Flannagan, R. S., Harrison, R. E., Yip, C. M., Jaqaman, K., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+macrophage+&amp;#34;probing&amp;#34;+is+required+for+the+efficient+capture+of+phagocytic+targets.">Dynamic macrophage &amp;#34;probing&amp;#34; is required for the efficient capture of phagocytic targets.</a> J. Cell Biol. 191, 1205-1218 (2010).</li><li>Swanson, J. 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The authors declare no conflicts of interest.

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 ...

visualizing-the-early-stages-of-phagocytosis

Research

JoVE Journal

Biology

16.5K Views.  Oregon Health & Science University. 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 µm including zymosan and IgG-coated beads.

Video: Visualizing the Early Stages of Phagocytosis

Layers of Symbiosis - Visualizing the Termite Hindgut Microbial Community

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth.  Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth. Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different ...

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and memory and learning4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR).&#160;This technique allows investigators to study early stages of development that were previously difficult to access.

The zebra finch (Taeniopygiaguttata) is a valuable model organism; however, early stages of zebra finch development have not been extensively studied. The protocol describes how to dissect early embryos for developmental and molecular applications.

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...