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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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Sphingolipid+Biosynthesis+Blocks+Phagocytosis+of+Candida+albicans.">Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.</a> PLoS Pathog. 11, e1005188 (2015).</li><li>Kwik, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+cholesterol,+lateral+mobility,+and+the+phosphatidylinositol+4,5-bisphosphate-dependent+organization+of+cell+actin.">Membrane cholesterol, lateral mobility, and the phosphatidylinositol 4,5-bisphosphate-dependent organization of cell actin.</a> PNAS. 100, 13964-13969 (2003).</li><li>Levin, R., Grinstein, S., Schlam, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoinositides+in+phagocytosis+and+macropinocytosis.">Phosphoinositides in phagocytosis and macropinocytosis.</a> BBA. 1851, 805-823 (2015).</li><li>Freeman, S. 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Cell. 21, 511-527 (2005).</li><li>Strijbis, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bruton's+Tyrosine+Kinase+(BTK)+and+Vav1+contribute+to+Dectin1-dependent+phagocytosis+of+Candida+albicans+in+macrophages.">Bruton's Tyrosine Kinase (BTK) and Vav1 contribute to Dectin1-dependent phagocytosis of Candida albicans in macrophages.</a> PLoS Pathog. 9, e1003446 (2013).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta-glucan+receptor+Dectin-1+activates+the+integrin+Mac-1+in+neutrophils+via+Vav+protein+signaling+to+promote+Candida+albicans+clearance.">The beta-glucan receptor Dectin-1 activates the integrin Mac-1 in neutrophils via Vav protein signaling to promote Candida albicans clearance.</a> Cell Host Microbe. 10, 603-615 (2011).</li><li>Esteban, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+recognition+is+mediated+by+the+association+of+dectin-1+and+galectin-3+in+macrophages.">Fungal recognition is mediated by the association of dectin-1 and galectin-3 in macrophages.</a> PNAS. 108, 14270-14275 (2011).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Meth. 9, 671-675 (2012).</li><li>Ettinger, A., Wittmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+live+cell+imaging.">Fluorescence live cell imaging.</a> Meth. Cell Biol. 123, 77-94 (2014).</li><li>Khodjakov, A., Rieder, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+division+process+in+living+tissue+culture+cells.">Imaging the division process in living tissue culture cells.</a> Methods. 38, 2-16 (2006).</li><li>Freeman, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+Form+an+Expanding+Diffusional+Barrier+that+Coordinates+Phagocytosis.">Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis.</a> Cell. 164, 128-140 (2016).</li><li>Jaumouille, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+cytoskeleton+reorganization+by+Syk+regulates+Fcgamma+receptor+responsiveness+by+increasing+its+lateral+mobility+and+clustering.">Actin cytoskeleton reorganization by Syk regulates Fcgamma receptor responsiveness by increasing its lateral mobility and clustering.</a> Dev. Cell. 29, 534-546 (2014).</li><li>Bain, J., Gow, N. A., Erwig, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+insights+into+host-fungal+pathogen+interactions+derived+from+live-cell+imaging.">Novel insights into host-fungal pathogen interactions derived from live-cell imaging.</a> Sem. Immunopath. 37, 131-139 (2015).</li><li>Forestier, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+host-Leishmania+interactions:+significance+in+visceral+leishmaniasis.">Imaging host-Leishmania interactions: significance in visceral leishmaniasis.</a> Para. Immunol. 35, 256-266 (2013).</li><li>John, B., Weninger, W., Hunter, C. 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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 ...

The over goal of this technique is to monitor the different stages of phagocytosis using a confocal microscope. This method can help answer key questions in the field of cross pathogens interactions. Mainly to understand how pathogens enter the host cells during infection.The main advantage of this technique is that it is simple and enable us to gain information about the virus cell of our events that occur during phagocytosis in a time sensitive manner. To begin, prepare the fluorescently conjugated zymosan as described in the accompanying text protocol. The previous day, seed about five times 10 to the fourth DC 2.4 cells in a chamber slide, so that the wells are 60 to 70%confluent at the time of the experiment.Once the cells are at a proper density, place the chamber slide at 10 degrees Celsius for 10 minutes. Cooling of the cells is necessary to block endocytosis as well as when to toggle cytosis. And subsequently to have a synchronized phagocytosis.Next gently remove the medium from each well using a pipette or aspirator. Then, mix the fluorescently conjugated zymosan with cell culture medium at a multiplicity of an infection of 10. Next, add 500 microliters of medium into each chamber well to ensure that all of the cell are covered.Then spin down the chamber slide to both increase the contacts between the particulates in the cells and synchronize the bonding process. Following centrifugation, transfer the chamber slide to a 37 degree Celsius humidified incubator with 5 percent CO2. After five minutes at 37 degrees Celsius, remove the chamber slide from the incubator and aspirate the medium.Then, wash the cells three times with ice cold PBS. After the final wash, check for the presence of unbound particles with a light microscope and perform additional washes if necessary. It's essential to remove unbound particles from the wells to minimize background noise during the analysis.Next, fix the cells by adding 500 microliters of four percent paraformaldehyde in PBS to the wells, and incubating them for 30 minutes at room temperature. After fixing the cells, wash them by adding 500 microliters of PBS into each chamber and then gently aspirating the solution. Repeat the washing step two more times.Following the third round of washing, stop the reaction by adding 500 microliters of 50 millimolar ammonium chloride in PBS to the cells and incubating them for 10 minutes. Then, wash the cells in 500 microliters of room temperature PBS. Next, permeabilize the cells by adding 500 microliters of binding buffer to each well and incubating the cells for 30 minutes at room temperature.The next step is to stain for F-actin by adding fluorescently conjugated phalloidin to the cells and incubating them for one hour at room temperature. Then wash the cells three times with 500 microliters of PBS. Finally, capture images using a confocal microscope and analyze the images using ImageJ.In order to measure the zymosan uptake, prepare chamber slides with cells as shown in the binding assay. Following the cooling and labeling steps, transfer the chamber slide to a 37 degree Celsius humidified incubator with five percent CO2. And incubate the cells for different time points.Fix the cells after 30, 60, and 90 minutes post infection. Be sure to optimize these time points based on the respective cell type and fluorescent particles that are used. Then, analyze images using ImageJ.Successful uptake is characterized by a complete internalization of a particulate inside the cytoplasm of a phagocyte. To begin, prepare a microscope stage environmental chamber by warming it to 37 degrees Celsius and equilibrating the chamber with 5 percent CO2. Wait 30 to 45 minutes for the temperature and the CO2 to stabilize before imaging.Seeing the CO2 and the temperature levels are critical for phagocytosis, as important to turn on the microscope heater prior to the experiment to equilibrate the environment of that chamber. Next, prepare chamber slides with DC 2.4 cells which stabilate express mCherry F-actin as shown in the binding assay. Following the cooing and labeling steps, transfer the chamber slide from the centrifuge directly onto a pre-warmed microscope stage.Finally, perform live imaging using a confocal laser scanning microscope as described in the accompanying text protocol, to not only capture the movement and dynamics of the F-actin network, but also to visualize the different phagocytic events within the living cells. The cells shown here in green is a wild type DC 2.4 cell that is being infected with Canadian BFP which is shown in red. Timelapse imaging is used to visualize the early stages of phagocytosis and is shown here in 30 second intervals.The locations denoted by the asterisk are where actin remodeling is occurring. The entire process occurs in a matter of a few minutes. Here the role of sphingolipids were evaluated for their role in the early stages of phagocytosis.Using this technique, sphingolipids were found to be essential for the binding of candida albicans to the cells and reduced overall phagocytosis. This reduction of phagocytotic binding was also shown for DC 2.4 cell binding to zymosans. After watching this video you should have a good understanding of how to perform live imaging to study the early stage of phagocytosis.Once mastered, this technique can be done in one to two hours if it is performed properly. Following this procedure, the phagocytosis of other pathogens, including bacteria and the parasites, can be performed in order to understand the intricate relationship between the immune cells and of this pathogens. After its development, this technique paved the way for researchers in field of cell biology, to explore receptor mediated endocytosis including phagocytosis and a macropinocytosis in immune cells.

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16.4K Views.  Oregon Health & Science University. The over goal of this technique is to monitor the different stages of phagocytosis using a confocal microscope. This method can help answer key questions in the field of cross pathogens interactions. Mainly to understand how pathogens enter the host cells during infection.The main advantage of this technique is that it is simple and enable us to gain information about the virus cell of our events that occur during phagocytosis in a time sensitive manner. To begin, prepare the fluoresce...

Video: 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...

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Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

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Accumulation and Distribution of Fluorescent Microplastics in the Early Life Stages of Zebrafish

Zebrafish embryos/larvae develop externally and are optically transparent. The bioaccumulation of microplastics in fish at early life stages is readily assessed with fluorescently labeled...

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Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput...

dissection and downstream analysis of zebra finch embryos at early stages of development

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

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.

non-invasive assay for chlorophyll biosynthesis kinetics determination during early stages of arabidopsis de-etiolation

Author Spotlight: Non-Invasive High-Resolution Measurement of Chlorophyll Synthesis During De-Etiolation

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

two-step approach to explore early- and late-stages of organ formation in the avian model: the thymus and parathyroid glands organogenesis paradigm

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

visualizing axonal growth cone collapse and early amyloid &#946; effects in cultured mouse neurons

Visualizing Axonal Growth Cone Collapse and Early Amyloid &amp;#946; Effects in Cultured Mouse Neurons

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented.  This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's...

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

visualizing the effects of a positive early experience, tactile stimulation, on dendritic morphology and synaptic connectivity with golgi-cox staining

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire...

macrophage cholesterol depletion and its effect on the phagocytosis of cryptococcus neoformans

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

phagocytosis assay for apoptotic cells in drosophila embryos

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

visualizing early infection sites of rice blast disease (magnaporthe oryzae) on barley (hordeum vulgare) using a basic microscope and a smartphone

Visualizing Early Infection Sites of Rice Blast Disease Magnaporthe oryzae on Barley Hordeum vulgare Using a Basic Microscope and a Smartphone

This is a straightforward protocol of a barley leaf sheath assay using minimal reagents and common laboratory equipment (including a basic smartphone). The purpose is to visualize the early infection process of blast disease in labs without access to advanced microscopy...

Visualizing Early Infection Sites of Rice Blast Disease (Magnaporthe oryzae) on Barley (Hordeum vulgare) Using a Basic Microscope and a Smartphone

assessing the cellular immune response of the fruit fly, drosophila melanogaster, using an in vivo phagocytosis assay

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

an assay to study the regulation of macrophage phagocytosis by mesenchymal stem cells

This video demonstrates an assay to study mesenchymal stem cell (MSC) regulation of macrophage phagocytosis in a co-culture. Upon co-culturing MSC with macrophages, non-opsonized zymosan particles conjugated to a pH-sensitive fluorophore are added to be phagocytosed by the macrophages. With the increase in phagocytosis, the fluorescence intensity rises, allowing the determination of MSC-mediated regulation of phagocytic...

An Assay to Study the Regulation of Macrophage Phagocytosis by Mesenchymal Stem Cells

phenotypic analysis of rodent malaria parasite asexual and sexual blood stages and mosquito stages

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria...

mesenchymal stem cell regulation of macrophage phagocytosis; quantitation and imaging

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent...

neutrophil phagocytosis assay

Neutrophil Phagocytosis Assay

live-cell video microscopy of fungal pathogen phagocytosis

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages.  These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of...

assessing the effects of lipofuscin-like granules on rpe phagocytosis: total consumptive capacity

Assessing the Effects of Lipofuscin-Like Granules on RPE Phagocytosis: Total Consumptive Capacity  

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 ...

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 ...

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 ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...

Isolation and Profiling of Single Nuclei from Frozen Mammalian Tissues

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

Author Spotlight: Enhancing Drug Discovery - Development of Automated, Standardized Protocols for Nuclei Extraction from Frozen Tissues 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.