The study was supported by grants from the Fondazione Bambino Ges&#249; and Ricerca Corrente (Italian Ministry of Health) to C.C.&#160;&#160;We would like to thank Dr Enrico Bertini (Department of Neuroscience, Unit of Neuromuscular and Neurodegenerative Diseases, Laboratory of Molecular Medicine, Bambino Ges&#249; Children&#39;s Research Hospital), Dr Stefania Petrini (Confocal Microscopy Core Facility, Research Laboratories, Bambino Ges&#249; Children&#39;s Research Hospital), Giulia Pericoli&#160;(Department of Onco-hematology, Gene and Cell Therapy, Children&#8217;s Research Hospital Bambino Ges&#249;) and Roberta Ferretti (Department of Onco-hematology, Gene and Cell Therapy, Children&#8217;s Research Hospital Bambino Ges&#249;) for scientific discussions and technical help. Maria Vinci is recipient of a &#8220;Children with Cancer UK fellowship&#8221;.

1. Coating 96 well plates
NOTE: Different coatings were tested in the same plate but separate wells (see Supplemental File).
<ol>
	<li>Dilute the Matrigel 1:100 in DMEM. Add 100 &#181;L per well to the 96 well plates and incubate for 1 h at room temperature. Following this, remove the solution and wash the wells with 100 &#160;&#181;L&#160;of DMEM twice.</li>
	<li>Dilute laminin (20 &#181;g/mL, LN-521) in PBS (with calcium and magnesium). Add 100 &#181;L to the well and incubate at 4 &#176;C overnight. The following day perform two washes with DMEM before seeding the cells.</li>
	<li>Dilute vitronectin (10 &#181;g/mL) in dilution buffer. Add 100 &#181;L per well to the 96 well plate and incubate for 1 hour at room temperature. Wash the wells with PBS (without calcium and magnesium) before plating the cells.</li>
	<li>Dilute HU-Fibronectin (30 &#181;g/mL) in ddH2O. Add 100 &#181;L to the wells and incubate at room temperature for 45 minutes. Following this, wash the wells with the medium before seeding the cells.</li>
</ol>
2. Maintenance of iPSCs in culture
NOTE: iPSCs were purchased commercially. The iPSCs were derived from healthy human fibroblasts and reprogrammed using episomal technology.
<ol>
	<li>From the -80 &#176;C freezer or liquid nitrogen, thaw the cryopreserved iPSCs in a 37 &#176;C water bath. Clean the vial containing the cells with 70% ethanol prior to moving it into the biological safety cabinet.</li>
	<li>Add the cell suspension to 5 mL of pre-warmed cell culture media (e.g., mTeSR1) drop by drop with a 1000 &#181;L pipette in a 15 mL sterile conical tube.</li>
	<li>Centrifuge the cells at 304 x g for 5 min at room temperature (RT).</li>
	<li>Remove the media and resuspend the cell pellet in 4 mL of cell culture medium.</li>
	<li>Plate the cell suspension into two wells of the 6 well plates (105 iPSCs per 6-well cell culture dish), where the mouse embryonic fibroblasts (MEFs) have been plated previously. Seed MEFs two days before plating iPSCs at a density of 2.4&#8201;x&#8201;104/cm2 in DMEM (containing 10% Fetal Bovine Serum, 1% L-glutamine and 1% Penicillin-Streptomycin).</li>
	<li>After seeding, supplement the cell media with 10 &#181;M of ROCK inhibitor Y-27632.</li>
	<li>Grow the iPSCs on MEFs for the first 4-5 weeks and then in feeder free condition (MEF free condition), using one of the coating of interest (see step 1 and Table 1) in mTeSR1.</li>
	<li>When the iPSCs are 70-80% confluent, passage 1:4 using 0.5 mM EDTA treatment for 3-5 min at RT. Add 1 mL of 0.5 mM EDTA for a 6 well plate (or proportional quantities for other types of plates). Transfer to new wells in feeder-free conditions and incubate at 37 &#176;C, 5% CO2, 20%&#160;O2.&#160;&#160;</li>
	<li>Change the media with fresh mTeSR1 every day and split the cells every 2 days.</li>
</ol>
3. Characterization of cell confluence
<ol>
	<li>Use 96 well plates for the experiments.</li>
	<li>Seed 10,000 cells per well following at least 1 month of culturing in feeder free condition in order to be sure that the MEFs were not passaged. Use disposable counting slides &#160;to count the cells with the optical microscope.</li>
	<li>Perform the experiments in triplicate. Therefore, test each seeding condition in three wells.</li>
	<li>Perform automated image acquisition from day 1 following seeding using a cytometer in bright-field mode. Perform automated image acquisition every 24 h for 5 days. For detailed information on the experimental parameters, refer to the Supplemental File.</li>
	<li>Use auto-contrast and auto-exposure to better visualize cells.</li>
	<li>Set the analysis setting (see Supplemental File) for confluence analysis to apply a mask of 60%, 80% and 100% per well, in order to evaluate the changes in focus due to the light refraction at the border of the wells. Use the different mask analysis setting mentioned above to analyze the cell confluence at each time point.</li>
</ol>
4. Statistical analyses
<ol>
	<li>Report quantitative results as means &#177; standard error of the mean (SEM).</li>
	<li>For comparing overall differences of the different coating conditions, obtain data using the same samples and perform the Student&#8217;s paired-sample t-test. P values less than 0.05 are considered statistically significant, and all reported p-values are two sided.</li>
</ol>
5. Characterization of the cytoskeletal microfilaments
<ol>
	<li>Fix cells with 4% paraformaldehyde (4% PFA) in PBS for 10 min at RT, followed by two washes in PBS (10 min total).</li>
	<li>Add 100 &#181;L of blocking solution (composed by 5% BSA, 0.1% Triton in PBS) to each well for 1 h at RT.</li>
	<li>Remove the blocking solution and wash the samples twice with PBS for 10 min.</li>
	<li>Add 100 &#181;L of the phalloidin-conjugate working solution per sample and incubate for 1 h at RT.</li>
	<li>Wash cells twice with PBS (10 min at RT).</li>
	<li>Stain nuclei with Hoechst 33342 diluted 1:10000 in PBS for 10 min at RT.</li>
	<li>Remove the Hoechst solution and wash the cells twice with PBS for 10 min each time.</li>
	<li>Wash the sample with H2O and let dry under a chemical hood.</li>
	<li>Add 100 &#181;L of mounting media (i.e. PBS:glycerol, 1:1) to cover the cells and preserve fluorescence of samples.</li>
	<li>Observe cell at Ex/Em 493/517 nm on a laser-scanning confocal microscope equipped with a white light laser (WLL) source and a 405 nm diode laser. Acquire sequential confocal images using a HC PLAPO 40x oil-immersion objective. Use the same laser power, beam splitters, filter settings, pinhole diameters and scan mode for all examined samples.</li>
</ol>

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The authors have nothing to disclose.

The use of iPSCs for disease modeling and future drug screening together with their possible application in precision medicine makes it a technology of great relevance and for this reason we believe that it is necessary to clearly understand the in vitro culturing condition that better resemble the physiological situation of embryonic stem cells. In this context, we tested different ECM coatings using&#160;wild type iPSCs in order to understand the conditions that allow the cells to remain in a healthy and undifferentiat...

Induced pluripotent stem cells (iPSCs) are obtained from somatic cells and can be differentiated into different cell types. They are often used as a system to model disease pathogenesis or perform drug screening, and also offer the potential to be used in the context of personalized medicine. Since iPSCs have great potential, it is important to fully characterize them for use as a reliable model system. We previously showed the importance of growing iPSCs in a hypoxic environment as these cells rely on glycolysis and an aerobic environment can cause redox imbalance1. iPSCs are also vulnerable to other culture conditions, particularly the extracellular environment. Optimization of culture conditions is a key issue to keep them healthy and proliferating. A healthy iPSC culture will lead to healthy differentiated cells that generally are the endpoint of the model used to understand molecular, cellular and functional features of specific human disorders or cellular processes.
In this study, a simple protocol has been used to test the confluence of iPSCs using different coating conditions in separate wells. iPSCs require a feeder layer of murine embryonic fibroblasts (MEF) in order to properly attach, but the coexistence of iPSCs and MEF makes it difficult to perform analysis like RNA or protein extraction since two populations of cells are present. In order to avoid the feeder layer, different proteins belonging to the extracellular matrix (ECM) have been used to recreate the natural cell niche and to have feeder free iPSC culture. In particular, Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, which is enriched in extracellular matrix proteins (i.e., laminin, collagen IV, heparan sulfate proteoglycans, entactin/nidogen, and growth factors)2,3. The other used coating conditions are instead purified proteins with known relevance in building the ECMs: laminin-521 is known to be secreted by human pluripotent stem cells (hPSCs) in the inner cell mass of the embryo and it is one of the most common laminins in the body after birth4,5,6,7,8,9,10,11; vitronectin is a xeno-free cell culture matrix known to support growth and differentiation of hPSC12,13,14,15,16; fibronectin is an ECM protein important for vertebrate development and the attachment and maintenance of embryonic stem cells in a pluripotent state17,18,19,20,21,22,23,24,25. Since different coating conditions are available, we compare them in terms of their effect on iPSCs&#8217; confluence.

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1123">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">10 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td style="width:281px;">Corning</td>
			<td style="width:77px;">4488</td>
			<td style="width:176px;">Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL high-clarity polypropylene (PP) conical centrifuge tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td>Tool</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (With Ca2+; Mg2+)</td>
			<td>Thermofisher</td>
			<td>14040133</td>
			<td>Medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (without Ca2+; Mg2+)</td>
			<td>Euroclone</td>
			<td>ECB4004L</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td>Corning</td>
			<td>4487</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture microplate, 96 WELL, PS, F-Bottom</td>
			<td>Greiner Bio One</td>
			<td>655090</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture plate, 6 well</td>
			<td>Costar</td>
			<td>3516</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM (Dulbecco&#39;s Modified Eagle&#39;s Medium- high glucose)</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>ED4SS-500g</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epi Episomal iPSC Reprogramming Kit</td>
			<td>Invitrogen</td>
			<td>A15960</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FAST - READ 102</td>
			<td>Biosigma</td>
			<td>BVS100</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Merck</td>
			<td>FC010</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>Reagent</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">H2O</td>
			<td></td>
			<td>MILLIQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst</td>
			<td>Thermofisher</td>
			<td>33342</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminin 521</td>
			<td>Stem Cell Technologies</td>
			<td>77003</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>LS25030081</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>Corning Matrigel hESC-Qualified Matrix</td>
			<td>354277</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">Mouse embryonic fibroblasts (MEF)</td>
			<td>Life Technologies</td>
			<td>A24903</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Medium</td>
			<td>Stem Cell Technologies</td>
			<td>85851</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Supplement</td>
			<td>Stem Cell Technologies</td>
			<td>85852</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vitronectin</td>
			<td>Stem Cell Technologies</td>
			<td>7180</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>Reagent</td>
		</tr>
	</tbody>
</table>

measuring the confluence of ipscs using an automated imaging system

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

In this study, we investigated iPSCs confluence when grown on different coating conditions. Using a cytometer, we were able to obtain readily informative results in triplicates in 5 days. Since iPSCs hardly attach to plastic vessels and a coating is necessary to support their proliferation, we decided to monitor the confluence of human iPSCs as it is indicative of the health of the cell culture and it may reflect on their differentiation potential. After in vitro expansion, we seeded the iPSCs on different ECM substrates...

Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

Measuring the Confluence of iPSCs Using an Automated Imaging System

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

measuring-the-confluence-of-ipscs-using-an-automated-imaging-system

Research

JoVE Journal

Biology

1.9K Views.  Ospedale Pediatrico Bambino Gesù, IRCCS. The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

Video: Measuring the Confluence of iPSCs Using an Automated Imaging System

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.