The study was supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

The described protocol was employed in accordance with NIH rules on the use of human tissue samples.
1. Tissue Preparation
<ol>
	<li>Prior to beginning, obtain human tissue specimens according to Institutional Review Board (IRB) protocols.</li>
	<li>Choose the type of tissue/cell block and corresponding processing method [formalin-fixed paraffin-embedded (FFPE), frozen, or ethanol-fixed paraffin-embedded (EFPE)]. Formalin fixation provides optimal histology, followed by ethanol fixation and flash frozen. However, fixation and tissue processing methods can affect DNA, RNA, and protein quantity and quality for downstream molecular analysis and should be considered.</li>
	<li>Cut the tissue/cell block sections onto the slide type (glass, membrane glass, or metal frame membrane) of choice.&#160;SIVQ analysis works equally well on all three slide types. Please note that the pseudo-coverslip method (described below) with xylenes and ethanol cannot be performed on the metal frame membrane slides since the slide must be inverted on the stage.</li>
	<li>Select a chemical- or IHC-based tissue stain to identify cells of interest from the background. Note that staining methods can also affect DNA, RNA, and protein quality and quantity of the tissue. Test the tissue after staining to assess baseline quality of biomolecules before proceeding with the protocol. SIVQ-LCM has been performed on tissue/cytology slides stained with: immunohistochemistry (IHC) with DAB15, immunofluorescence, fast red, de novo red, toluidine blue, and hematoxylin and eosin (H&#38;E) (unpublished data).</li>
</ol>
2. Specimen Imaging
<ol>
	<li>Load slides onto the motorized stage of the microdissection instrument and launch the related software. Select the check boxes to designate the positions of the loaded slides and ensure the captured image files are to be saved in jpeg format.</li>
	<li>Optimize the image quality by adjusting the brightness and focus of the image on the screen, using either the manual focus wheel or through the dissection instrument&#8217;s software.
	<ol>
		<li>Using the Image Toolbox within the dissection instrument&#8217;s software, set the lamp brightness and camera gain appropriately. Example values are brightness = 60 and gain = 220, with the diffuser.</li>
		<li>Focus either manually or with the autofocus feature in the software.</li>
	</ol>
	</li>
	<li>Capture a thumbnail overview image of the slide to provide a roadmap for the dissection process.
	<ol>
		<li>For optimal image quality of an uncoverslipped slide, utilize the diffuser below the condenser on the instrument, or, add a small amount (~30 &#956;l) of either ethanol or xylenes to improve the refractory index (pseudo-coverslip). When using xylenes, be aware they are toxic and proper safety measures must be used, including the use of a fume hood and protective lab coat, eye goggles, and gloves.</li>
		<li>Do NOT place the LCM cap on the slide until the ethanol or xylenes solution has completely cleared or the polymer on the cap will be distorted.</li>
	</ol>
	</li>
	<li>Navigate the slide and capture images of the areas to be dissected at 10X, 20X, or 40X magnification. If necessary, enhance the image with software such as AutoCorrect (in Microsoft Office Picture Manager) as previously described22.&#160;Images must be captured in jpeg format to allow them to be re-imported into the automated selection software.</li>
</ol>
3. Algorithm Analysis of the Image
<ol>
	<li>Transfer captured images from the microdissection instrument to the SIVQ folder.&#160;Install and open the ArcturusXT, AutoScan and SIVQ software packages on the computer attached to the dissection instrument. For access to the SIVQ software, please contact Dr. Ulysses Balis (ulysses@med.umich.edu).</li>
	<li>Open SIVQ and load the captured image (jpeg) of interest.</li>
	<li>Navigate to the area of interest and/or adjust the size of the display windows (Viewport 5 &#38; 6).&#160;In the SIVQ software, Viewport 5 shows the pre-processing image and Viewport 6 shows the post-processing image16.</li>
	<li>Choose the size of the ring vector and number of rings to be used.</li>
	<li>Select the predicate image feature to be captured by right clicking on it in Viewport 6.</li>
	<li>Click &#8220;scan&#8221; to analyze the image.</li>
	<li>Adjust the statistical probability of image matching using the two sliding bars.&#160;The upper bar adjusts the overall vector specificity, and is used to exclude area from the initial scan (with sensitivity as defined by the selected &#8220;Stat&#8221; variable) that may represent excessive included area.&#160;Conversely, the lower slider is utilized to increase sensitivity, after a scan is performed, with the intent of increasing area that is classified as being a match.&#160;Both of these slider controls utilize the &#8220;Stat&#8221; variable as the initial baseline sensitivity threshold.</li>
	<li>To save the image, click &#8220;save as jpeg&#8221; (image is now saved in c:/vq_test folder/pics).</li>
	<li>Analyze the image with the algorithm. The output of the algorithm analysis needs to result in an annotated image for it to be used in SIVQ-LCM. At present, the current version of the SIVQ core engine is in beta version testing with the expectation that the full production version (available Q1 2014) will incorporate a full software development kit (SDK) and application programming interface (API) for simplified integration of user-generated spatial filters and downstream workflow data processing steps with the core ring matching engine.&#160;This SDK will be distributed with a complete set of documentation.
	<ol>
		<li>Ensure that the SIVQ heatmap is changed to a uniform red color.</li>
	</ol>
	</li>
	<li>Export the image. It is essential to re-embed the positional coordinates in the post-analysis jpeg image by using a HEX editor to paste in the file header from the original dissection instrument image.&#160;The appropriate data may be found between &#8220;Start of Image&#8221; marker (0xFF, 0xD8) and the first&#160;&#8220;Define Quantization Table&#8221; marker (0xFF, 0xDB).</li>
</ol>
4. Microdissection
<ol>
	<li>Place the LCM cap at the center of the region of interest, where the images were captured for the SIVQ analysis.</li>
	<li>Calibrate and Quality Control (QC) the UV/IR lasers and optimize the parameters, including power, duration, laser locations, and UV cutting speed (as recommended by the manufacturer). Perform these calibrations before re-importing the analyzed image.&#160;</li>
	<li>Open AutoScanXT (automated selection software) and import the analyzed image from c:/vq_test folder/pics.</li>
	<li>Train the automated selection software to recognize the SIVQ annotation and create the dissection map.
	<ol>
		<li>To create a training file, select four regions of interest (marked by &#8220;blue circles&#8221;) on the &#8220;red paint&#8221; of the SIVQ analyzed image.</li>
		<li>Select the background areas (marked by &#8220;red squares&#8221;) that are not to be dissected.</li>
		<li>Click on the &#8220;Analyze&#8221; button to generate the training file, which can be saved for subsequent uses.&#160;</li>
	</ol>
	</li>
	<li>Perform microdissection using the appropriate infrared (IR) and/or ultraviolet (UV) lasers.
	<ol>
		<li>Copy the selected areas onto the &#8220;live&#8221; image.</li>
		<li>In the &#8220;microdissect&#8221; toolbox, select the appropriate IR capture or UV cutting buttons.</li>
	</ol>
	</li>
	<li>After the dissection is complete, move the LCM cap to the QC station and capture an image of the dissected tissue/cells. Another approach is to place the cap on a blank area of the slide prior to moving it to the QC station, this allows the user to take pictures at various magnifications.</li>
	<li>Capture an image of the tissue area of interest after microdissection to further assess lifting efficiency.</li>
	<li>If the desired cells have been dissected successfully, click on the &#34;present stage&#34; button in the ArcturusXT software and remove the LCM cap to initiate the molecular extraction procedure for downstream analysis.</li>
</ol>

<ol>
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B., 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=Gene+expression+in+Barrett's+esophagus:+laser+capture+versus+whole+tissue.">Gene expression in Barrett's esophagus: laser capture versus whole tissue.</a> Scandinavian journal of gastroenterology. 44, 787-795 (2009).</li><li>Espina, 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=Laser-capture+microdissection.">Laser-capture microdissection.</a> Nature. 1, 586-603 (2006).</li><li>Harrell, J. C., Dye, W. W., Harvell, D. M., Sartorius, C. A., Horwitz, K. 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W., 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=Impact+of+sample+acquisition+and+linear+amplification+on+gene+expression+profiling+of+lung+adenocarcinoma:+laser+capture+micro-dissection+cell-sampling+versus+bulk+tissue-sampling.">Impact of sample acquisition and linear amplification on gene expression profiling of lung adenocarcinoma: laser capture micro-dissection cell-sampling versus bulk tissue-sampling.</a> BMC medical genomics. 2, 13 (2009).</li><li>Zheng, J., Garg, S., Wang, J., Loose, D. S., Hauer-Jensen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissected+mucosa+versus+whole+tissue+specimens+for+assessment+of+radiation-induced+dynamic+molecular+and+pathway+changes+in+the+small+intestine.">Laser capture microdissected mucosa versus whole tissue specimens for assessment of radiation-induced dynamic molecular and pathway changes in the small intestine.</a> PloS one. 8, e53711 (2013).</li><li>Boone, D. R., Sell, S. L., Hellmich, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+of+enriched+populations+of+neurons+or+single+neurons+for+gene+expression+analysis+after+traumatic+brain+injury.">Laser capture microdissection of enriched populations of neurons or single neurons for gene expression analysis after traumatic brain injury.</a> J Vis Exp. (74), (2013).</li><li>Iyer, E. P., Cox, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+of+Drosophila+peripheral+neurons.">Laser capture microdissection of Drosophila peripheral neurons.</a> J Vis Exp. (39), (2010).</li><li>Hipp, 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=SIVQ-aided+laser+capture+microdissection:+A+tool+for+high-throughput+expression+profiling.">SIVQ-aided laser capture microdissection: A tool for high-throughput expression profiling.</a> Journal of pathology informatics. 2, 19 (2011).</li><li>Hipp, J. D., Cheng, J. Y., Toner, M., Tompkins, R. G., Balis, U. 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Michael R. Emmert-Buck is an inventor on NIH-held patents covering laser capture microdissection and receives royalty-based payments through the NIH Technology Transfer Program.

We present a protocol for the application of SIVQ-LCM to microdissect immunostained epithelial cells from FFPE human breast tissue. The use of an image analysis algorithm, such as SIVQ, reduces the amount of hands-on time required for the microdissection process. This is a potentially important advance for the field since operator time and effort is typically the rate-limiting step for the precise dissection of cells of interest. In the present protocol, we specifically adapted our procedure to the ArcturusXT instrument,...

Originally developed in the mid-1990s, laser capture microdissection (LCM) enables the user to precisely capture specific cells or cellular regions from a histological tissue section via microscopic visualization1,2. Many studies comparing molecular analysis of LCM versus tissue scrapes illustrate the value of the method3-12. In addition, there are three video protocol publications on the technology that are available for viewing13,14. However, despite its proven value, LCM can be tedious and laborious when the target of interest is a dispersed cell population in a heterogeneous tissue section, or when large numbers of cells are required for specific downstream applications such as proteomics. The burden placed on the human operator led us to develop a semi-automated dissection approach for LCM by combining a powerful image analysis algorithm to guide the LCM process15.
In collaboration with the University of Michigan, our laboratory at the NIH extended the previously developed and reported Spatially invariant vector quantization (SIVQ) algorithm in a manner to allow it to semi-automate the tissue selection process intrinsic to guided microdissection, thus making available a tool with the pathologist or life scientist in mind. Spatially invariant vector quantization (SIVQ) is an algorithm that allows the user to simply &#8220;click&#8221; on a histological feature of interest to create a ring vector (predicate image feature) that can be used to search the entire histological image, adjusting the statistical threshold as needed16-21. The resultant heat map displays the quality of matches to the initial predicate image feature and is subsequently converted into a single color (red) annotation map that can be imported into the LCM instrument. The automated selection software, AutoScanXT, is then used to draw a map based on SIVQ&#39;s annotation guiding the capture of the target cells from the tissue sample. The detailed protocol below describes the implementation of SIVQ into the microdissection workflow.

<table border="0" cellpadding="0" cellspacing="0" width="586">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Positive Charged Glass Slides</td>
			<td style="width:84px;">Thermo Scientific</td>
			<td style="width:119px;">4951Plus-001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Xylenes, ACS reagent, &#8805;98.5% xylenes + ethylbenzene basis&#160;</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">247642</td>
			<td style="width:167px;">CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Ethyl Alcohol, U.S.P. 200 Proof, Anhydrous</td>
			<td style="width:84px;">The Warner-Graham Company</td>
			<td style="width:119px;">6.505E+12</td>
			<td style="width:167px;">CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Arcturus CapSure Macro LCM Caps</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">LCM0211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">ArcturusXT Laser Microdissection Instrument</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">ARCTURUSXT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:216px;">AutoScanXT Software</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">An optional image analysis program for the ArcturusXT Laser Microdissection Device. This is software is required for SIVQ-LCM.</td>
		</tr>
		<tr height="169">
			<td height="169" style="height:169px;width:216px;">Spatially Invariant Vector Quantization (SIVQ)</td>
			<td style="width:84px;">University of Michigan</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">This tool suite is publicly available for academic collaborations. For access to the SIVQ algorithm, please contact Dr. Ulysses Balis [Ulysses@med.umich.edu]</td>
		</tr>
	</tbody>
</table>

sivq-lcm protocol for the arcturusxt instrument

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.

A FFPE human breast tissue section was immunostained for cytokeratin AE1/AE3 using a standard IHC protocol23. After staining, the tissue slide was placed on the ArcturusXT stage and the SIVQ-LCM protocol was initiated as described above. Since the tissue cannot be coverslipped for microdissection, the IHC+ stained cells can be difficult to discern visually (Figure 1A). Thus, to provide better index matching and an improved image, xylenes were added to the tissue section to create a pseudo-cove...

Watch this Scientific Journal Video about SIVQ-LCM Protocol for the ArcturusXT Instrument at JoVE.com

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

sivq-lcm-protocol-for-the-arcturusxt-instrument

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JoVE Journal

Biology

8.5K Views.  National Institutes of Health. SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

Video: SIVQ-LCM Protocol for the ArcturusXT Instrument

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called thigmomorphogenesis. In past decades, several signaling components have been identified and reported for being involved in the mechanotransduction (e.g., calcium ion binding proteins and jasmonic acid biosynthesis enzymes). However, the relatively slow pace of research in the study of force signaling or thigmomorphogenesis is largely attributed to two reasons: the requirement for laborious human hand-manipulated touch induction of thigmomorphogenesis and the force strength errors associated with people&#8217;s hand-touch. To enhance the efficiency of external force loading on a plant organism, an automatic touch-force loading machine was built. This robotic arm-driven hair brush touches provide a labor-saving and easily repeatable touch-force simulation, unlimited rounds of touch repetition and adjustable touch strength. This hair touch-force loading machine can be used for both large scale screening of touch-force signaling mutants and the phenomics study of plant thigmomorphogenesis. In addition, touch materials such as human hair, can be replaced with other natural materials like animal hair, silk threads and cotton fibers. The automated moving arms on the machine may be equipped with water sprinkling nozzles and air blowers to mimic the natural forces of rain drops and wind, respectively. By using this automatic hair touch-force loading machine in combination with the hand-performed cotton swab touching, we have investigated the touch response of two force signaling mutants, MAP KINASE KINASE 1 (MKK1) and MKK2 plants. The phenomes of the touch-force loaded wild type plants and two mutants were evaluated statistically. They have exhibited significant differences in touch response.

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...

Quantifying Mitophagy in C. elegans and a Liver Cancer Cell Line

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. 
Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

Author Spotlight: Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes  

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...