Name: isolation of viable multicellular glands from tissue of the carnivorous plant, nepenthes
Uploaded: 2013-12-22T20:30:00
Description: Watch this Scientific Journal Video about Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes at JoVE.com

We thank the greenhouse team at the MPI-CE at the Botanical Garden of the Friedrich Schiller University, Jena, for cultivating plants and Wilhelm Boland and the Max Planck Society for support.

1. Plant Material Preparation
<ol>
	<li>All Nepenthes species were grown either in the greenhouses of the MPI for Chemical Ecology or the Botanical Garden in Jena, Germany.</li>
	<li>For the preparation of secreting tissue harvest a pitcher and remove the upper one third including lid, peristome, and slippery zone. The pitcher can be used either freshly or frozen in liquid nitrogen and stored at -80 &#176;C.</li>
</ol>
2. Microdissection of Pitcher Tissue
<ol>
	<li>The microsampling p...

<ol>
	<li>Adlassnig, W., Peroutka, M., Land, I., Lichtscheidl, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glands+of+carnivorous+plants+as+a+model+system+in+cell+biological+research.">Glands of carnivorous plants as a model system in cell biological research.</a> Acta Bot. Gall. 152, 111-124 (2005).</li><li>Darwin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insectivorous plants. , (1875).</li><li>Mith&#246;fer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carnivorous+pitcher+plants:+Insights+in+an+old+topic.">Carnivorous pitcher plants: Insights in an old topic.</a> Phytochemistry. 72, 1678-1682 (2011).</li><li>Owen Jr., T. P., Lennon, K. A., Santo, M. J., Anderson, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+for+nutrient+transport+in+the+pitchers+of+the+carnivorous+plant.">Pathways for nutrient transport in the pitchers of the carnivorous plant.</a> Nepenthes alata Ann. Bot. 84, 459-466 (1999).</li><li>Brandt, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microgenomics:+gene+expression+analysis+at+the+tissue-specific+and+single-cell+levels.">Microgenomics: gene expression analysis at the tissue-specific and single-cell levels.</a> J. Exp. Bot. 56, 495-505 (2005).</li><li>H&#246;lscher, D., Schneider, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+laser-assisted+microdissection+for+tissue+and+cell-specific+analysis+of+RNA,+proteins,+and+metabolites.">Application of laser-assisted microdissection for tissue and cell-specific analysis of RNA, proteins, and metabolites.</a> Prog. Bot. 69, 141-167 (2008).</li><li>Rottloff, S., M&#252;ller, U., Kilper, R., Mith&#246;fer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropreparation+of+single+secretory+glands+from+the+carnivorous+plant+Nepenthes.">Micropreparation of single secretory glands from the carnivorous plant Nepenthes.</a> Anal. Biochem. 394, 135-137 (2009).</li><li>Liu, W. H., Saint, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+quantitative+method+of+real+time+reverse+transcription+polymerase+chain+reaction+assay+based+on+simulation+of+polymerase+chain+reaction+kinetics.">A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics.</a> Anal. Biochem. 302, 52-59 (2002).</li><li>Werker, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trichome+diversity+and+development.">Trichome diversity and development.</a> Adv. Bot. Res. Inc. Adv. Plant Pathol. 31, 1-35 (2000).</li><li>Fahn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+secretory+cells.">Structure and function of secretory cells.</a> Adv. Bot. Res. Inc. Adv. Plant Pathol. 31, 37-75 (2000).</li><li>Juniper, B. E., Robins, R. J., Joel, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The carnivorous plants. , (1989).</li><li>Gershenzon, J., McCaskill, D., Rajaonarivony, J., Mihaliak, C., Karp, F., Croteau, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+secretory-cells+from+plant+glandular+trichomes+and+their+use+in+biosynthetic-studies+of+monoterpenes+and+other+gland+products.">Isolation of secretory-cells from plant glandular trichomes and their use in biosynthetic-studies of monoterpenes and other gland products.</a> Anal. Biochem. 200, 130-138 (1992).</li><li>Rontein, D., Onillon, S., 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=CYP725A4+from+yew+catalyzes+complex+structural+rearrangement+of+taxa-4(5),11(12)-diene+into+the+cyclic+ether+5(12)-oxa-3(11)-cyclotaxane.">CYP725A4 from yew catalyzes complex structural rearrangement of taxa-4(5),11(12)-diene into the cyclic ether 5(12)-oxa-3(11)-cyclotaxane.</a> J. of Biol. Chem. 283 (5), 6067-6075 (2008).</li><li>Aharoni, A., Jongsma, M. A., Bouwmeester, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+science?+Engineering+of+terpenoid+biosynthesis+in+plants.">Volatile science? Engineering of terpenoid biosynthesis in plants.</a> Trends Plant Sci. 10, 594-602 (2005).</li><li>Mith&#246;fer, A., Boland, W., Maffei, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+ecology+of+plant-insect+interactions.">Chemical ecology of plant-insect interactions.</a> Ann. Plant Rev. 34, 261-291 (2009).</li><li>Kennedy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+pests,+parasitoids,+and+predators:+Tritrophic+interactions+involving+the+genus+Lycopersicon.">Tomato pests, parasitoids, and predators: Tritrophic interactions involving the genus Lycopersicon.</a> Ann. Rev. Entomol. 48, 51-72 (2003).</li><li>Mith&#246;fer, A., Boland, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+defense+against+herbivores:+Chemical+aspects.">Plant defense against herbivores: Chemical aspects.</a> Ann. Rev. Plant Biol. 63, 431-450 (2012).</li><li>Rottloff, S., Stieber, R., Maischak, H., Turini, F. G., Heubl, G., Mith&#246;fer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+a+class+III+acid+endochitinase+from+the+traps+of+the+carnivorous+pitcher+plant+genus,+Nepenthes.">Functional characterization of a class III acid endochitinase from the traps of the carnivorous pitcher plant genus, Nepenthes.</a> J. Exp. Bot. 62, 4649-4647 (2011).</li></ol>

For a long time, studies of plant secretory structures, in particular trichomes have been performed essentially on anatomy and were purely descriptive. There is a large body of publications of this type as summarized in reviews by9-11. Our biochemical and molecular knowledge of plant secretory structures is still limited in spite of some early remarkable work e.g. on mint glandular trichomes showing that glandular trichomes are the site of biosynthesis of monoterpenoids which are secreted into the sub...

Plants produce a wide huge variety of compounds with different industrial and pharmaceutical applications, ranging from small molecular weight molecules to polymers. Such compounds are frequently produced and secreted by highly specialized structures, many of which are localized at the surface of the plants such as glandular trichomes or nectaries, or internally such as idioblasts or latex and resin ducts, respectively. However, relatively little is known about the biology of these specialized secretory structures, although all types of those glands display many features that are indicative of active metabolism. In particular, glands of carnivorous plants such as Nepenthes can be seen as model systems in plant cell biology1.
Carnivorous plants are the object of several studies since 1875 when Charles Darwin&#39;s &#39;Insectivorous Plants&#39; was published2. However, only in the last few years research on the molecular level has been done and still our knowledge is limited. For example, the protein composition of the digestion fluid in the pitcher plants of the genus Nepenthes is still not completely known. Only recently several hydrolytic proteins have been identified and a few of the corresponding genes as well3. In the pitfall-traps of Nepenthes (Figure 1), multicellular glands are localized at the inner bottom of the pitchers. These glands do both production and secretion of the digestive enzymes into the pitcher fluid and absorption of dissolved nutrients from the pitcher fluid4. Hence, these bifunctional glands represent a unique microtissue with a central position and function in the carnivory of Nepenthes that is worth to be studied in detail.
<img alt="Figure 1" src="/files/ftp_upload/50993/50993fig1.jpg" /> 
Figure 1. Nepenthes alata pitcher. The lower part of the pitcher contains the digestive fluid and glands on the inside.
<a href="http://www.jove.com/files/ftp_upload/50993/50993fig1highres.jpg" target="_blank">Click here to view larger image</a>.
In plant sciences, many experimental approaches rely on &#39;bulk material&#39; because no defined tissue is investigated but entire organs. As a consequence, their homogenization and analysis can only provide results that are diluted and proportionately with respect to individual tissues and cells5. To solve this problem, micropreparation techniques, for example the so-called laser capture microdissection (LCM), were developed and successfully used to harvest specific plant tissues and analyze their individual contents6. However, LCM is often limited if unstable and/or fast degrading cell components are targeted such as RNA. In such a case, the need of previous tissue fixation that is time consuming and often accompanied by degradation of the biomolecules, is of disadvantage. Moreover, due to the high water content of many plant tissues and the strength of cell walls, LCM often failed to prepare straight from fresh tissue7. Any approach to investigate cellular components that are present in secretory glands from genes, mRNA, and proteins to secondary compounds and their biosynthesis needs alternative techniques for the particular preparation. Thus, specific techniques to isolate single cells or even multicellular glands that remain intact after preparation are necessary.
Here a method is presented, employing a mechanized microsampling platform that features a direct microscopic visualization. This technique is both rapid and efficient in the preparation of individual glands from the pitchers of Nepenthes species. These multicellular glands are bifunctional, i.e. they are involved in secretion as well as in uptake processes, they are sessile, and not exposed cell layers are impregnated to form an endodermis. Entire glands can be isolated without neighboring tissue and deposited in a well-defined manner into a PCR tube. This technique is applied to study specific genes as well as gene expression in the glands&#39; tissues.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Name of Material/ Equipment</td>
		</tr>
		<tr>
			<td>RNAqueous-Micro Kit</td>
			<td>Life Technologies GmbH, Germany</td>
			<td>AM1931</td>
			<td>Isolation of total RNA, 50 samples</td>
		</tr>
		<tr>
			<td>Dynabeads mRNA DIRECT Micro Kit</td>
			<td>Life Technologies GmbH, Germany</td>
			<td>61011</td>
			<td>5 ml Dynabeads Oligo (dT)25</td>
		</tr>
		<tr>
			<td>DynaMag-2 magnet</td>
			<td>Life Technologies GmbH, Germany</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript CellsDirect cDNA Synthesis System</td>
			<td>Life Technologies GmbH, Germany</td>
			<td>18080-200</td>
			<td>25 reactions</td>
		</tr>
		<tr>
			<td>RNaseOUT</td>
			<td>Life Technologies GmbH, Germany</td>
			<td>10777-019</td>
			<td>Rnase inhibitor, 5,000 units</td>
		</tr>
		<tr>
			<td>Random Hexamers (0.4 &#956;g/&#956;l)</td>
			<td>QIAGEN GmbH, Germany</td>
			<td>79236</td>
			<td>100 &#956;l</td>
		</tr>
		<tr>
			<td>10 mM dNTP Mix, molecular biology grade</td>
			<td>Fermentas GmbH, Germany</td>
			<td>R0192</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Taq DNA polymerase, (recombinant)</td>
			<td>Fermentas GmbH, Germany</td>
			<td>EP0402</td>
			<td>Supplied with 10x Taq buffer (with KCl or (NH4)2SO4) and 25 mM MgCl2</td>
		</tr>
		<tr>
			<td>TURBO DNase</td>
			<td>Life Technologies GmbH, Germany</td>
			<td>AM2238</td>
			<td>1,000 units</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Carl Roth GmbH Co. Germany</td>
			<td>T846.3</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneRuler Low Range DNA ladder, ready-to-use</td>
			<td>Fermentas GmbH, Germany</td>
			<td>SM1193</td>
			<td>50 &#956;g</td>
		</tr>
		<tr>
			<td>Brilliant II SYBR Green QPCR Master Mix</td>
			<td>Agilent Technologies, Inc., USA</td>
			<td>600828</td>
			<td>Single kit</td>
		</tr>
		<tr>
			<td>RNaseZAP</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>R2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Carl Roth GmbH &#38; Co., Germany</td>
			<td>CK08.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Carl Roth GmbH &#38; Co., Germany</td>
			<td>2801.1 / 2855.1</td>
			<td></td>
		</tr>
		<tr>
			<td>GE NanoVue spectrophotometer</td>
			<td>GE Healthcare Europe GmbH, Germany</td>
			<td>28-9569-62</td>
			<td></td>
		</tr>
		<tr>
			<td>Mastercycler gradient</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>5331 000.010</td>
			<td></td>
		</tr>
		<tr>
			<td>Mx3000P Real-Time PCR System</td>
			<td>Stratagene, USA</td>
			<td>401403</td>
			<td></td>
		</tr>
		<tr>
			<td>Agagel Standard Horizontal Gel Electrophoresis Chamber</td>
			<td>Biometra</td>
			<td>Discontinued</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>aura optik gmbh</td>
			<td>aureka</td>
			<td>Equipped with a microforceps</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Carl Zeiss Microscopy, Jena, Germany</td>
			<td>SteREO Lumar.V12</td>
			<td>Equipped with an AxioCam</td>
		</tr>
		<tr>
			<td>Lumar Filter set 01</td>
			<td>Carl Zeiss, Microscopy, Jena, Germany</td>
			<td>485001-0000-000</td>
			<td>UV filter</td>
		</tr>
		<tr>
			<td>Lumar Filter set 09</td>
			<td>Carl Zeiss, Microscopy, Jena, Germany</td>
			<td>485009-0000-000</td>
			<td>non-UV filter</td>
		</tr>
	</tbody>
</table>

isolation of viable multicellular glands from tissue of the carnivorous plant, nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

For the preparation of single glands from Nepenthes pitcher tissue, a mechanized microdissection technique was developed and applied. The micropreparation platform needs to be equipped with microforceps, attached to the micromanipulator, which in turn are operated by a control panel. Individual glands, visible under the microscope, could be targeted with the forceps that grasped the gland tissue and removed it mechanically (Figure 4A).
The glandular tissue was transfe...

Watch this Scientific Journal Video about Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes at JoVE.com

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

isolation-viable-multicellular-glands-from-tissue-carnivorous-plant

Research

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Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37&deg;C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent   a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37&deg;C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.

This procedure describes the detection and isolation of mouse TH17 leukocytes that actively secrete IL-17 upon stimulation.

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Here we describe an efficient strategy to remove the silk-producing glands from the abdomen of female black widow spiders. This procedure allows the rapid isolation of the seven distinct silk-producing glands in a highly purified fashion, an important process for investigators studying spider silk production and fiber assembly.

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and the Plasmodium parasites that cause malaria, accumulate in the secretory cavities of SG cells. Here, they are poised for transmission to their vertebrate hosts during a subsequent blood meal. As adult glands form as an elaboration of larval SG duct bud remnants that persist beyond early pupal SG histolysis, the larval SG is an ideal target for interventions that limit disease transmission. Understanding larval SG development can help develop a better understanding of its morphology and functional adaptations and aid in the assessment of new interventions that target this organ. This video protocol demonstrates an efficient technique for isolating, fixing, and staining larval SGs from Anopheles gambiae mosquitoes. Glands dissected from larvae in a 25% ethanol solution are fixed in a methanol-glacial acetic acid mixture, followed by a cold acetone wash. After a few rinses in phosphate-buffered saline (PBS), SGs can be stained with a broad array of marker dyes and/or antisera against SG-expressed proteins. This method for larval SG isolation could also be used to collect tissue for in situ hybridization analysis, other transcriptomic applications, and proteomic studies.

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

The adult mosquito salivary gland (SG) is required for the transmission of all mosquito-borne pathogens to their human hosts, including viruses and parasites. This video demonstrates efficient isolation of the SGs from the larval (L4) stage Anopheles gambiae mosquitoes and preparation of the L4 SGs for further analysis.

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and ...

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria

Plastoglobule lipid droplets are a dynamic sub-compartment of plant chloroplasts and cyanobacteria. Found ubiquitously among photosynthetic species, they are believed to serve a central role in the adaptation and remodeling of the thylakoid membrane under rapidly changing environmental conditions. The capacity to isolate plastoglobules of high purity has greatly facilitated their study through proteomic, lipidomic, and other methodologies. With plastoglobules of high purity and yield, it is possible to investigate their lipid and protein composition, enzymatic activity, and protein topology, among other possible molecular characteristics. This article presents a rapid and effective protocol for the isolation of plastoglobules from chloroplasts of plant leaf tissue and presents methodological variations for the isolation of plastoglobules and related lipid droplet structures from maize leaves, the desiccated leaf tissue of the resurrection plant, Eragrostis nindensis, and the cyanobacterium, Synechocystis sp. PCC 6803. Isolation relies on the low density of these lipid-rich particles, which facilitates their purification by sucrose density flotation. This methodology will prove valuable in the study of plastoglobules from diverse species.

A fast and efficient protocol is presented for the isolation of plastoglobule lipid droplets associated with various photosynthetic organisms. The successful preparation of isolated plastoglobules is a crucial first step that precedes detailed molecular investigations such as proteomic and lipidomic analyses.

Plastoglobule lipid droplets are a dynamic sub-compartment of plant chloroplasts and cyanobacteria. Found ubiquitously among photosynthetic species, they ...