This study is supported by the grant of National Social Science Foundation (22FYSB023), Hubei Industrial Design Center Research Foundation (08hqt201412046), and Humanities and Social Science Foundation of Hubei Provincial Education Department (15Y054).

Full written informed consent was provided by all participants. Ethics approval was not applicable since no animal or human tissue samples were included in the present study.
1. Investigating the requirements of the user community
<ol>
	<li>Deliver questionnaires to the biologists/experimenter working on cell wound healing assays. Collect the completed questionnaires and use them for the study. For this study, from 12 Oct 2021 to 3 Feb 2022, 100 questionnaires were delivered to 100 biologists. The response percent was 97%.</li>
	<li>Ask respondents to answer questions like, which of the cell scratching experiments bothered you the most? and which tool was used to perform the cell scratching? Follow these questions with sub-questions to trace the causes.</li>
	<li>Investigate the experimentalists&#39; perceptions of tools such as pipette tips and cell culture inserts in cell wound healing experiments. Understand and gather their reasons for selecting one tool over another, using the Means-end-chain theory based on laddering techniques in the questionnaires section14,15. 
	&#8203;NOTE: The ladder method is divided into two types: the soft ladder method with in-depth interviews and the hard ladder method with questionnaires or paper-and-pencil quizzes16. The hard laddering method was the primary technique utilized in this study. The Means-end-chain theory suggests that the explicit knowledge held by the target users is superficial and concrete, while the tacit knowledge is deep and abstract17,18,19.</li>
</ol>
2. Design and three-dimensional modeling
<ol>
	<li>Draw a sketch from the insights gathered from the previous questionnaire survey and initiate the design process. Use this preliminary sketch as the foundational blueprint.
	<ol>
		<li>Elaborate on the dimensions and layout of each component by applying the Dim, DimRadius, and DimDiameter functions in the modeling software.</li>
		<li>Perform measurements by the vernier caliper with a precision of 0.02 mm on actual 6-well plates to ascertain the final dimensions of the cell scraper. Confirm these to be 42.1 mm in length, 42.1 mm in width, and 18.5 mm in height. Pay attention to the details in the design phase, especially the product height and fitness in the well, to facilitate a smoother assembly.</li>
	</ol>
	</li>
	<li>Construct a three-dimensional model and render it.
	<ol>
		<li>Begin 3D designing in a software by creating a basic model. Click buttons like ExtrudeCrv for stretching and Loft for shaping the design. Refine the model and then click FilletEdge for smooth edges. 
		NOTE: Other used function buttons include, Lines - To draw straight line segments, Polylines - To create continuous lines composed of multiple segments, Rectangles - To draw rectangular shapes, Circles - To draw circular shapes, Arcs - To draw arc or elliptical shapes, Points - To place single point markers, Text - To add text labels, Dimensions - To add measured dimensions to models, Array - To create copied patterns of objects, Rotate - To rotate objects to desired angles, Move - To move objects to new locations, Scale - To resize objects bigger or smaller, Trim/Extend - To trim or extend objects to meet other geometry, Boolean Union/Difference/Intersection - To combine, subtract from, or find the intersection of objects, Layers - To organize objects on different layers, Render - To generate rendered views with materials and lighting and Export - To export model geometry to other file formats.</li>
		<li>Import the model into a 3D rendering software. Apply materials including plastic, sponge, and steel, then adjust the lighting for realistic rendering. 
		NOTE: A generalized list of function buttons include, Drag &#38; Drop - To apply materials directly to parts or models by dragging a material from the library and dropping it onto the desired component in the real-time view, Right-click - When right-clicking on a material in the library, you will typically see options to: Apply to Selection - Apply the material to a selected part in the real-time view. Edit Material - Adjust the material properties. Material Properties (after selecting a material) - To access and modify specific properties of the material, such as color, roughness, refractive index, and other attributes. Search Bar - To quickly find a material in the library by typing its name or associated keywords. Categories/Folders - To navigate through different categories or folders of materials like metals, plastics, glass, etc. Add to Favorites - To mark certain materials as favorites for easy access during future sessions. Hotkeys - Some operations can be accessed via hotkeys. For instance, M is usually the hotkey to quickly bring up the material properties of a selected part.</li>
		<li>Finally, enhance the image&#39;s contrast (+56) and saturation (Vibrance +19, Saturation +7) in photo and design software, and add background elements (text information and color from white to light gray gradient background) for context to ensure a high-quality, realistic product representation.
		<ol>
			<li>Use Image &#62; Adjustments, Brightness/Contrast to adjust the contrast of an image. Use Image &#62; Hue/Saturation to modify the saturation level of the image. Use Text Tool (T icon) to add text information to the image . 
			&#8203;NOTE: A generalized list of function buttons include Ellipse Tool (U) - To draw dots/circles. Set the tool to draw a shape, choose the fill color you want, and then click and drag (holding Shift to maintain a perfect circle) to create a dot. Line Tool (U) - To draw straight lines on the image. Select the tool, set the desired line width, and then click and drag to draw a line. Brush Tool (B) - An alternative way to draw dots and lines. Select the brush size and shape, then click (for dots) or click and drag (for lines).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After completing the three-dimensional model, proceed to produce the cell scraper via the grinding and assembly method20,21,22.</li>
</ol>
3. Production
<ol>
	<li>Employ the color, materials, finish (CMF) theory in the current study to design the prototypes of the cell scraper. CMF theory serves as a design methodology in scientific research. It enhances product usability, shapes user perception, and directs the selection of materials23,24,25.
	<ol>
		<li>Select the colors to be used in the cell scraper from standard color system, including Black 6 C, Cool Gray 6 C, and 11-0601TPG26,27.</li>
		<li>To build the cell scraper that meets lab standards, choose appropriate materials including polypropylene (PP), sponge, and high-carbon steel. For the fabrication of physical prototypes, initiate by employing a 3D printer to produce the structural framework. Subsequently, utilize connectors or adhesive agents to complete assembling the scraper. 
		&#8203;NOTE: To prepare the physical product of the cell scraper, grind and assemble the necessary materials. Safety protocols must be followed throughout the entire process to ensure a safe and effective final product.</li>
		<li>Move on to the finishing process, which may involve cutting, grinding, polishing, stamping, or other techniques. Start by sanding the model using a medium-grit, 120-grit sandpaper to remove any rough edges.</li>
		<li>Follow this with a fine-grit, 220-grit sandpaper to achieve a smooth surface.</li>
		<li>Post-sanding to smooth out the surfaces, ensuring the plug-in or built-in connectors are correctly matched.</li>
		<li>Join slide I and II securely using the connectors (fixed rods) to ensure a robust and durable assembly of the cell scraper. Achieve the final fixed form of the cell scraper through a combination of mechanical fastening and adhesive bonding. Use fasteners, specifically a mortise and tenon structure, to secure parts together through mechanical force. Additionally, to enhance the assembly&#39;s strength, apply adhesive (glue) to bond the components further.</li>
		<li>Autoclave the scraper at 121.3 &#176;C for 30 min to ensure it retains its original shape and physical properties.</li>
	</ol>
	</li>
</ol>
4. Cell culture
<ol>
	<li>Grow HOS cells in minimum essential medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Culture them at 37 &#176;C with 5% CO2.</li>
	<li>Change the culture medium every 2 days.</li>
	<li>When cell growth surpasses 80% confluency, add 1 mL of trypsin with 0.25% EDTA and digest for 60 s. Afterwards, centrifuge the cells at 300 x g for 5 min for subculture. Remove supernatant and add culture medium to get a final concentration of 5 x 104 cells per well. Use an automated cell counter for counting cells.</li>
</ol>
5. Assessing cell wounding potential of the cell scraper and tips-based method
<ol>
	<li>Prepare all materials for wounding and sterilize them with ultraviolet radiation by exposing for 30 min.</li>
	<li>After sterilization, wound 100% confluent HOS cells in 6-well plates at a concentration of 5 x 104 cells per well by using either the cell scraper method or the tips-based method.
	<ol>
		<li>For the tip-based method, use a 100 &#181;L tip and drag it across the cell containing medium with 1 stroke in the horizontal direction and the other in the vertical direction.</li>
		<li>For the cell scraper method, place the scraper prototype in one of the wells and with the help of the tweezer gently press once. The cells are wounded. Carry out each wounding experiment in triplicate.</li>
	</ol>
	</li>
	<li>Analyze all cell wounds using a digital microscope system and imaging software.</li>
</ol>
6. Measuring cell viability and cell proliferation
<ol>
	<li>Perform all cell viability assays following the protocol provided in the CCK-8 Kit.</li>
	<li>Incubate the cells with CCK-8 solution for 2 h at 37 &#176;C, then measure their absorbance at 450 nm using a microplate reader to quantify cell viabilities.</li>
	<li>Design the 5-ethynyl-20-deoxyuridine (EdU) incorporation assay to accurately quantify DNA duplication and directly quantify the cell proliferation ratio. Determine the influence of different methods to generate cell wounds upon cell proliferation using the EdU assay, following the protocol outlined in previous publication28.</li>
	<li>Stain the cells and image them using a digital microscope system. Ensure that each experiment is carried out in triplicate.</li>
</ol>

Innovating an Improved Cell Scraper for Cell Wounding by User Requirements Analysis

The authors declare that they have no conflict of interest.

The present study aimed to develop an automatic mechanized tool for cell wound healing assay. To the best of our knowledge, it represents the first attempt to apply a mechanized driven structure to create cell wounds in a one-click way automatically. Through this, we aim to address the shortcomings of the traditional tips-based method, such as low reproducibility and unstable scratch state. Benefiting from the positive results and the encouraging feedback from the user community, the cell scraper is expected to provide e...

Tumors are characterized by distinct hallmarks such as selective growth advantages, metabolic rewiring, and immune modulation, all of which intriguingly contribute to enhanced cell migration, a critical malignant behavior of tumor cells. This feature directly affects the distant metastasis of the primary tumor, compromising the long-term survival of patients1,2,3. Selective growth advantages enable cancer cells to outcompete normal cells, while metabolic rewiring supports this rapid proliferation by altering energy pathways. Concurrently, immune modulation allows tumors to evade the body&#39;s defenses. Studies underscore the severity of this issue, showing that lung metastasis, often a result of enhanced cell migration, is a terminal event leading to the death of patients with various cancers4. For instance, breast cancers5, cervical carcinoma4, and osteosarcoma6 account for 20%, 9%, and 30% of such cases, respectively. Therefore, assessing tumor cell migration has become integral to current oncology research, further highlighting the multifaceted nature of tumor progression.
Cell wound healing assay is an easy-to-use method for measuring in vitro cellular migration, often employed in oncological studies7. Most experimenters use pipette tips to create cell wounds manually8. Although such a method could form cell wounds rapidly and conveniently, it still has many limitations that affect reproducibility and accuracy for evaluating cell migration. Firstly, using pipette tips to create scratches manually is highly influenced by the operator&#39;s operating angle, force, and speed, affecting the method&#39;s repeatability8. Secondly, tip-generated cell defects usually have jagged edges rather than straight edges because pipette tips are plastic products with certain elasticity9. Some studies generate wounds by placing prefabricated culture inserts directly into the cell culture plate to restrict the range of cell proliferation10. This approach circumvents the limitation of the tips-based method, such as jagged edges and reproducibility. However, even with biocompatible materials, the long-term coexistence of the embedding with the cells still impacts cell growth11. Moreover, the embedding may also cause cellular epigenetic changes in the marginal zone due to contact restriction12. Also, contact-insert produced from biocompatible materials are expensive and difficult to reuse, limiting their feasibility13. Therefore, a novel, reproducible, and practical tool is needed to easily quantify in vitro cellular migration.
The primary goal of this method is to introduce an innovative tool for quantifying in vitro cellular migration in oncological studies, addressing the limitations of existing techniques and enhancing reproducibility and accuracy in assessing cellular migration.
The rationale behind this technique&#39;s development lies in the critical importance of evaluating tumor cell migration in oncology. Tumors exhibit distinctive hallmarks, including selective growth advantages, metabolic rewiring, and immune modulation, all contributing to enhanced cell migration, a fundamental aspect of cancer malignancy. This method aims to provide a more dependable means of studying cellular migration, contributing to a deeper understanding of tumor behavior.
This method offers substantial advantages over existing techniques. Manual scratch assays can suffer from operator-dependent interference, while culture inserts may impact cell growth and gene expression. In contrast, this method offers improved repeatability, accuracy, and practicality, presenting a cost-effective solution for measuring in vitro cellular migration in oncology research. It addresses a crucial need for a reliable and accessible tool to study cell migration in various cancer types, making it a valuable addition to the field.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CCK-8 Kit</td>
			<td>Beyotime Company, China</td>
			<td>C0037</td>
			<td></td>
		</tr>
		<tr>
			<td>digital microscope system</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Gibco, USA</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>HOS&#160;</td>
			<td>Procell Life Science &#38; Technology Co., Ltd</td>
			<td>CL-0360</td>
			<td></td>
		</tr>
		<tr>
			<td>Image-Pro Plus</td>
			<td>Media Cybernetics</td>
			<td>version 6.0</td>
			<td></td>
		</tr>
		<tr>
			<td>KeyShot</td>
			<td>Luxion</td>
			<td>version 11.0</td>
			<td>3D rendering software</td>
		</tr>
		<tr>
			<td>microplate reader</td>
			<td>BioTek, German</td>
			<td>ELX808</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>Gibco, USA</td>
			<td>11095080</td>
			<td></td>
		</tr>
		<tr>
			<td>Pantone matching system</td>
			<td>Pantone</td>
			<td></td>
			<td>commercial color matching</td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Beyotime Company, China</td>
			<td>ST488</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop</td>
			<td>Adobe</td>
			<td></td>
			<td>photo and design software</td>
		</tr>
		<tr>
			<td>Rhinoceros 3D</td>
			<td>Robert McNeel &#38; Associates</td>
			<td>version 7.0</td>
			<td>3D design software</td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter</td>
			<td>Bio-Rad</td>
			<td>TC20</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Cytiva&#160;HyClone, United State</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
	</tbody>
</table>

a tool to automatically create stable and reproducible cell-free gaps for improving the reliability of cell wound healing assay

The reliability of cellular migration measurement in wound healing assays is frequently undermined by the prevalent methodological instability, i.e., tip-based method. We introduce an innovative instrument designed to address these limitations. Our novel cell scraper surpasses the current approach, generating a more consistent and stable cell-free gap. Repeated biological experiments reveal that the cell-free gap produced by the cell scraper exhibits straighter edges and uniform size and shape compared to the tip-based technique (p &lt; 0.05). In terms of product design, the cell scraper boasts a refined color scheme suited for laboratory environments, enhancing the monitoring of experimental outcomes, and permits sterilization through autoclaving for reuse. Notably, after treatment, the cell scraper demonstrates a negligible effect on cell viability and proliferation (97.31% and 24.41%, respectively). Conversely, the tip-based method yields lower cell viability (91.37%) and proliferation (18.79%). This investigation presents the cell scraper as a novel, reusable device capable of generating reproducible cell-free gaps while preserving cellular viability, thereby augmenting the reliability of wound healing assays in comparison to existing techniques.

Dissecting user demands for tools to generate cell wounds 
The current experimental method to generate cell wounds demands further enhancement to address many issues that compromise biological reproducibility, robustness, economic consumption, and user experience of cell wound healing assay. We utilized the hard laddering method to analyze the requirements of users involved in biological experiments via questionnaires29 (Figure 1A). The informati...

A Tool to Automatically Create Stable and Reproducible Cell-free Gaps for Improving the Reliability of Cell Wound Healing Assay

Through the incorporation of interaction experience design and user requirements analysis, we introduce an innovative cell scraper that enhances cellular wound healing assays in terms of reproducibility, dependability, practicality, cellular integrity, and user experience.

The reliability of cellular migration measurement in wound healing assays is frequently undermined by the prevalent methodological instability, i.e., ...

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Biology

356 Views.  Kunming University of Science and Technology. Through the incorporation of interaction experience design and user requirements analysis, we introduce an innovative cell scraper that enhances cellular wound healing assays in terms of reproducibility, dependability, practicality, cellular integrity, and user experience.

Video: A Tool to Automatically Create Stable and Reproducible Cell-free Gaps for Improving the Reliability of Cell Wound Healing Assay

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...