The authors thank the Ellison Medical Foundation for their support. We wish also to thank members of the Abby Dernburg laboratory for assistance with using the wormsorter.

1. Obtaining a Synchronized Culture of Young Adult Animals
<ol>
	<li>Grow worms on several NGM plates seeded with OP50-1 E. coli&#160;bacteria (10x&#160;concentrated from a saturated culture) until many worms have reached the gravid stage.</li>
	<li>Treat gravid animals with egg-prep solution to obtain a synchronized culture (eggs).</li>
	<li>Plate eggs on several 60-mm NGM plates seeded with 10x&#160;concentrated OP50-1 at a density of roughly 150-200 eggs/plate.</li>
	<li>Incubate plates at 25&#160;&#176;C f...

<ol>
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The method we describe takes advantage of fluorescent labeling of entities outside of the worm, to follow interactions between the worm and its environment. In the case we present, separation was based on labeling of a pathogen and was employed to separate worms with heavy pathogen load from those with no (or light) load. Subsequent gene expression analysis found no difference in immune responses between the two groups suggesting that they were independent of pathogen load. The signal that initiates the response was show...

Automatic worm sorting is much like FACS, operating by measuring a fluorescent signal&#160;in a worm (typically provided by transgenic expression of reporter proteins) as it passes straightened in a tube, allowing redirection either to a collection tube/well or to a waste container&#160;according to gating parameters set by the researcher1. The wormsorter can facilitate research in many ways; an example of employing it as an analytical tool is a study that followed spatiotemporal patterns of promoter activity for almost 1,000 genes2. 
However, the major use of the wormsorter is in genetic screens, following target gene expression levels or localization of fluorescent protein along the axis of the worm3-5.
Here, we describe a new application for the wormsorter, in following colonization of the worm by a fluorescently tagged pathogen. With this as a tool we focused on the relationship between pathogen colonization/load and the immune response, to gain new insights into the mechanisms responsible for initiation of immune responses in the worm.
In virtually all organisms studied to date, initiation of innate immune responses to microbial pathogens depends on recognition of pathogen-associated molecular patterns (PAMPs), and/or danger/damage-associated molecular patterns (DAMPs)6,7. The first are conserved microbial structures that include components of the microbial cell wall, its flagellum, or its lipid bilayer6; the second, include both released molecules (e.g. ATP8), altered proteins or other markers of altered cellular processes9,10. Both types of signals are recognized by proteins designated as pattern recognition receptors (PRRs), which upon specific binding of a pattern molecule activate a chain of events leading to a protective response. C. elegans has been extremely useful as a tractable model to dissect various aspects of host-pathogen interactions, but one thing that is not well understood is how immune responses are initiated in the worm. None of the putative receptors that are orthologous to pattern recognition receptors (PRRs) in other organisms have been shown to bind PAMPs, and many of the orthologs of PRRs that are pivotal for immune responses in other organisms show a surprisingly limited contribution to worm pathogen responses and resistance. For example, the Drosophila&#160;Toll receptor, which is essential for resisting Gram positive pathogens, is represented in C. elegans by a sole homolog, tol-1, which contributes to protection from the Gram negative pathogen Salmonella Typhimurium11, but not from other tested Gram-negative, or -positive pathogens11,12 . These observations, combined with data indicating that immune responses could be induced by disrupting cellular protein translation has led some to suggest that C. elegans primarily detects DAMPs9,13,14. Nevertheless, reports describing ability of dead pathogens to induce immune responses suggest that PAMP binding may be have an important role in pathogen recognition in C. elegans15,16. Previous work focusing on immune responses in age-synchronized genetically identical C. elegans populations, demonstrated large individual variability in intestinal colonization by the bacterial Gram-negative pathogen Pseudomonas aeruginosa.
However, transcriptional profiling studies treated these variably-colonized populations as one entity17,18. Taking advantage of this variability, we developed a protocol focusing an automated wormsorter to separate differentially colonized populations of Caenorhabditis elegans exposed to GFP-expressing P. aeruginosa. Examining gene expression in differently-colonized populations facilitated assessment of the relationship between pathogen load (and the associated damage) and immune responses and provided new insights about pathogen recognition in C. elegans19. Below we describe the protocol, which could be applied to sort worms infected with any fluorescently labeled pathogen.
To potential users it should be noted that the number of worms required to be sorted out depends on the nature of the subsequent analyses and protocols in use. For example, in the case of microarray gene expression analysis, &#62;1,000 worms will be required to obtain enough RNA, if standard protocols are used, but ~100 worms would suffice if amplification is employed, allowing fast collection of material and thus minimizing stress to the worms.

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">M9 Buffer</td>
			<td style="width:139px;">Prepared in house</td>
			<td style="width:527px;">Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rifampicin&#160;</td>
			<td>Sigma</td>
			<td>R3501</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Egg prep solution&#160;</td>
			<td>Prepared in house</td>
			<td>50ml water ; 40ml bleach ; 10ml of 10N Sodium Hydroxide&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NGM plates&#160;</td>
			<td>Prepared in house</td>
			<td>Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SKP plates</td>
			<td>Prepared in house</td>
			<td>Recipe same as NGM only 0.35% peptone instead of 0.25%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Control test particles&#160;</td>
			<td>Union Biometrica&#160;</td>
			<td>310-5071-001</td>
		</tr>
	</tbody>
</table>

automated separation of c. elegans variably colonized by a bacterial pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

When age-matched, genetically identical C. elegans are exposed to P. aeruginosa, a wide distribution is observed in levels of colonization (Figure 1A). With the help of the protocol described here efficient separation of noncolonized from colonized worms can be achieved (Figure&#160;1B). Unlike noncolonized worms, colonized worms show signs of damage, such as sluggishness and reduced defecation19. The latter may be a reason why worms colonized by P. aerug...

Watch this Scientific Journal Video about Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen at JoVE.com

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

automated-separation-c-elegans-variably-colonized-bacterial

Research

JoVE Journal

Immunology and Infection

8.3K visualizações.  University of California, Berkeley.  O wormsorter facilita telas genéticas em Caenorhabditis elegans, classificando vermes de acordo com a expressão de repórteres fluorescentes. Aqui, descrevemos um novo uso: triagem de acordo com a colonização por um patógeno expressando GFP, e empregá-lo para examinar o papel mal compreendida de reconhecimento do patógeno em iniciar respostas imunes. 