This research was funded by CSIC I+D 2018, grant No. 95 (Mariana Sotelo Silveira).; CSIC Grupos (Omar Borsani) and PEDECIBA.

1. Preparation of the plant material and growth conditions
<ol>
	<li>To generate the needed plant material, sterilize the seeds of Arabidopsis wild type and mutant lines of interest. 
	NOTE: In this protocol, we used the following: ttl1: T-DNA insertion lines Salk_063943 (for TTL1; AT1G53300) - Columbia-0 (Col-0) wild-type; the Procuste1 (prc1-1) mutant, which consists of a knock-out mutation (Q720stop) in the CESA6 gene (AT5G64740), as previously described in10,11; and ttl1 x prc1-1 double mutant.
	<ol>
		<li>Put an approximate volume of 50 &#956;L of seeds in a microtube. Add 500 &#956;L of 70% ethanol + 0.01% Tween solution at room temperature. Mix and incubate for 7 min.</li>
		<li>Pour the ethanol solution and add 500 &#956;L of 20% sodium hypochlorite. Mix and incubate for 7 min.</li>
		<li>Pour the sodium hypochlorite solution and wash the seeds three to four times with sterile water.</li>
	</ol>
	</li>
	<li>Plating
	<ol>
		<li>Prepare in advance basal Murashige and Skoog (MS) medium12 + 1.5% sucrose + 1% agar (pH 5.7) (see Table of Materials). Autoclave the medium. Prepare a laminar flow hood to work in a sterile environment and label the square Petri dishes needed for the experiment. Pour the medium into the Petri dishes and allow the medium to solidify.</li>
		<li>Plate the sterilized seeds using sterile pipette tips onto square Petri dishes containing the medium. Distribute the seeds evenly in two rows. When the seeds are dry, close the lids and seal the plates. Stratify the seeds at 4 &#176;C in the dark for 4 days. 
		NOTE: Stratification is needed to synchronize germination.</li>
		<li>Place the plates vertically in the growth chamber in a long-day regime (16 h of light/8 h of dark) with 25 &#956;mol m2 s&#8722;1 light intensity and 23 &#176;C (day/night). Grow the seedlings for 7 days.</li>
	</ol>
	</li>
</ol>
2. Osmotic stress treatment (optional)
NOTE: This section provides details on the growth of Arabidopsis&#160;roots in osmotic potential of -1.2 MPa as estimated by cryoscopic osmometer (Table of Materials). This part can be omitted or changed depending on the experimental question at hand.
<ol>
	<li>Prepare basal MS medium + 1.5% sucrose + 400 mM mannitol + 1% agar (pH 5.7). Autoclave the medium. Pour the medium in square Petri dishes in the laminar flow hood and allow the medium to solidify.</li>
	<li>With tweezers (Table of Materials), place 5-day old seedlings pre-grown in basal MS + 1.5% sucrose medium in square Petri dishes containing the medium supplemented with 400 mM mannitol. Place the plates vertically in the growth chamber in the same conditions as in step 1.2.3. Grow the seedlings under this osmotic stress condition for 7 days. 
	NOTE: To evaluate root growth adaptation of the primary root during severe osmotic stress, it is suggested to use seedlings 5 days after germination grown in controlled conditions to ensure that the root meristem size is already established13. Alternatively, the analysis can be performed at every stage of development to determine the developmental effect of osmotic stress action.</li>
</ol>
3. Atomic force microscopy (AFM) nanoindentation experiments
<ol>
	<li>Preparation of the sample for nanoindentation experiments 
	NOTE: This is a crucial step for applying AFM to the study of live biological samples. The sample must be firmly attached to the substrate to be mechanically stable during indentation measurements. At the same time, sample structural qualities should be preserved. An effective strategy to stabilize a big sample for AFM is to use adhesives. The adhesive must dry quickly and must not be toxic or reactive with the surrounding medium. In this protocol, polystyrene Petri dishes were used as the substrate and non-acidic silicone glue (Table of Materials) was used to bind the Arabidopsis seedlings.
	<ol>
		<li>Spread a thin layer of silicone glue into the Petri dish with a coverslip. Leave the glue in the air for 45 s.</li>
		<li>With tweezers, place the seedling on the glue, orienting it in a direction that avoids contact between the seedling&#39;s protruding parts and the cantilever. Then, press the root gently to the silicone glue layer to bind it firmly. Leave the seedling in contact with the glue for 45 s before continuing with the next steps of the protocol.</li>
		<li>Incubate the attached seedlings with 2 &#956;g/mL propidium iodide (PI; Table of Materials) for 5 min in dark and carefully wash them thrice with 1x phosphate-buffered saline (PBS) solution (Table of Materials).</li>
		<li>Place the PI-incubated seedlings into an inverted fluorescence microscope coupled with the AFM. To observe fluorescence, set the excitation and emission wavelengths at 572 nm and 617 nm, respectively. The absence of fluorescence inside the epidermal cells confirms the viability of the roots.</li>
		<li>Cover the seedling with 1x phosphate-buffered saline (PBS) solution.</li>
	</ol>
	</li>
	<li>Indentation protocol 
	NOTE: Perform all AFM measurements within 1 h after the immobilization of the sample on its support at room temperature. The room where the AFM is located should be maintained at 25 &#176;C with an air conditioner. The PBS solution should be at the same temperature.
	<ol>
		<li>Mount a standard silicon nitride cantilever with a pyramidal tip (see Table of Materials) into the AFM probe holder for fluid (Table of Materials).</li>
		<li>Align the laser on the cantilever close to the position of the tip. Next, move the photodiode to place the laser spot at the center of the detector.</li>
		<li>To calibrate the deflection sensitivity, position a polystyrene Petri dish with 1x PBS under the AFM. 
		NOTE: This step is needed to calculate the indentation. When a force measurement is made on a hard surface, there is no indentation into the surface, so any change in the z scanner displacement corresponds to the cantilever deflection. Therefore, it is very important to calibrate the deflection sensitivity on a hard surface. Using a soft surface will overestimate the deflection sensitivity, which will result in spring constants that are too low.</li>
		<li>Adjust the photodetector such that the non-contact deflection is near 0 V. Calibrate the deflection sensitivity by performing an indentation (force curve), with a ramp size of 3 &#956;m, an indentation-and-retraction rate of 0.6 &#956;m/s, and a trigger threshold of 0.5 V.</li>
		<li>To calibrate the spring constant of the cantilever, use the Thermal Tune utility of the AFM software (Table of Materials) recommended for probes with spring constants less than approximately 5 N/m or use the method described in reference14. Before activating Thermal Tune in the software, ensure that the probe does not interact with the sample.
		<ol>
			<li>Enter the cantilever temperature. Click on Calibrate &#62; Thermal Tune or on the Thermal Tune icon in the NanoScope toolbar. Select a frequency range (1-100 Hz).</li>
			<li>Click on Acquire Data in the Thermal Tune panel. Wait for the AFM to acquire data, and then click on the Simple Harmonic Oscillator (Fluid) button.</li>
			<li>Adjust Median Filter Width to 3. Adjust PSD Bin Width to reduce the noise in the acquired data by averaging. Set fit boundaries around the first resonance peak.</li>
			<li>Click on Calculate Spring Constant k, and then, click on Yes in the pop-up window asking if the user wants to use this value.</li>
			<li>Repeat the steps described above three times and manually take an average of spring constant values. Enter this average value in the Spring Constant box in the Ramp parameter list in the PicoForce view. The calibration ends at this point.</li>
		</ol>
		</li>
		<li>Using the inverted optical microscope at 10x, 20x, and 40x eyepiece magnification, position the AFM probe on the surface of the fourth elongated epidermal cell of the primary root, ensuring to position it in the center of the cell.</li>
		<li>With the calculated spring constant value (step 3.2.5.5), obtain force curves with a ramp size of 3 &#181;m, a trigger threshold of 11 nN, and an indentation-and-retraction rate of 0.6 &#956;m/s at selected points. 
		&#8203;NOTE: Previous work15,16 found that low-frequency of indentation-and-retraction helps to minimize hysteresis and drag force.</li>
		<li>Obtain force curves from three cells per root for each treatment (use three biological replicates per treatment). Capture at least 150 force curves for each root.</li>
	</ol>
	</li>
</ol>
4. Measure the apparent Young&#39;s modulus
<ol>
	<li>Fit each force curve to the following model for pyramidal indenters17: <img alt="Equation 1" src="/files/ftp_upload/63533/63533eq01.jpg" style="margin: auto;" />, where E is the apparent Young&#39;s modulus of the sample, <img alt="Equation 2" src="/files/ftp_upload/63533/63533eq02.jpg" style="margin: auto;" /> is the Poisson modulus of the sample, and &#945; is the semi-angle to the vertex. Discard the fits with a correlation coefficient (r2) &#60; 0.99. Consider the Poisson&#39;s ratio for totally incompressible material: <img alt="Equation 2" src="/files/ftp_upload/63533/63533eq02.jpg" style="margin: auto;" /> = 0.5 and the geometry of the indenter given by the manufacturers. 
	NOTE: Use the first 100 nm from the contact point of the loading curve for the fitting to be as sensitive as possible to cell wall mechanics and to reduce the impact of turgor pressure on the apparent Young&#39;s modulus values8. The fitting has been done using a custom MATLAB program (available upon personal request by writing to J. C. B) that allows setting a specific indentation depth from the contact point of the loading curve.</li>
	<li>Create a normalized histogram with the apparent Young&#39;s modulus data and fit it with a Gaussian distribution. Discard data points outside the 95% confidence interval and recalculate both the histogram and the Gaussian fit. Calculate and report the mean and standard deviation of the apparent Young&#39;s modulus.</li>
	<li>Determine the significance of the comparisons between groups by multiple comparison tests like ANOVA.</li>
</ol>

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K., 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=Fifteen+compelling+open+questions+in+plant+cell+biology.">Fifteen compelling open questions in plant cell biology.</a> The Plant Cell. 34 (1), 72-102 (2022).</li><li>Zhang, B., Gao, Y., Zhang, L., Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plant+cell+wall:+Biosynthesis,+construction,+and+functions.">The plant cell wall: Biosynthesis, construction, and functions.</a> Journal of Integrative Plant Biology. 63 (1), 251-272 (2021).</li><li>Hamant, O., Haswell, E. 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(89), e51317 (2014).</li><li>Peaucelle, A., 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=Pectin-induced+changes+in+cell+wall+mechanics+underlie+organ+initiation+in+Arabidopsis.">Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis.</a> Current Biology. 21 (20), 1720-1726 (2011).</li><li>Fernandes, A. N., 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=Mechanical+properties+of+epidermal+cells+of+whole+living+roots+of+Arabidopsis+thaliana:+An+atomic+force+microscopy+study.">Mechanical properties of epidermal cells of whole living roots of Arabidopsis thaliana: An atomic force microscopy study.</a> Physical Review E. 85 (2), 21916 (2012).</li></ol>

The authors have no conflicts of interest. The MATLAB script used for fitting the data is available upon personal request by writing to J. C. B.

Cell and cell-wall mechanics are increasingly becoming relevant to gain insight into how mechanics affects growth processes. As physical forces propagate over considerable distances in solid tissues, the study of changes in the physical properties of the cell wall and how they are sensed, controlled, tuned, and impact the plant&#39;s growth are becoming an important field of study2,3,8.
A method is pr...

Plant cells are surrounded by a cell wall that is a complex structure composed of interacting networks of polysaccharides, proteins, metabolites, and water that varies in thickness from 0.1 to several &#181;m depending on the cell type and the phase of growth1,2. Cell wall mechanical properties play an essential role in the growth of plants. Low stiffness values of the cell wall have been proposed as a precondition for cell growth and cell-wall expansion, and there is increasing evidence that all cells sense mechanical forces to perform their functions. However, it is still debated whether changes in the physical properties of the cell wall determines cell fate2,3,4. Because plant cells do not move during development, the final shape of an organ depends on how far and in what direction a cell expands. Thus, Arabidopsis root is a good model to study the impact of cell wall physical properties in cell expansion because different types of expansion occur in different regions of the root. For example, anisotropic expansion is evident in the elongation zone and particularly noticeably in the epidermal cells5.
The method described here was used to characterize the physical properties of the cell wall of epidermal cells at the nanoscale of living Arabidopsis roots using an Atomic Force Microscope (AFM) coupled with an inverted fluorescence phase microscope6. For an extensive revision of the AFM technique, read7,8,9.
This protocol outlines a basic sample preparation method and a general method for AFM-based elasticity measurements of plant cell walls.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63533/63533fig01v2.jpg" /> 
Figure 1: Schematic overview of force-indentation experiment in Arabidopsis roots using atomic force microscopy (AFM). The scheme gives an overview of the steps of a Force-Indentation experiment from the preparation of the substrate to immobilize the root sample firmly (1-2), root viability confirmation through propidium iodide staining (3), cantilever positioning on the surface of an elongated epidermal cell of the primary root (4-5), force curves measurement (6), and force curve processing to calculate the apparent Young&#39;s modulus (7-8). EZ: elongation zone. <a href="https://www.jove.com/files/ftp_upload/63533/63533fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 x Phosphate-Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td>Include sodium chloride and phosphate buffer and is formulated to prevent osmotic shock and maintain water balance in living cells.</td>
		</tr>
		<tr>
			<td>AFM software</td>
			<td>Bruker, Billerica, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic force microscopy (AFM)</td>
			<td>BioScope Catalyst, Bruker, Billerica, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Catalyst Probe holder-fluid</td>
			<td>Bruker, Billerica, MA, USA</td>
			<td>CAT-FCH</td>
			<td>A probe holder for the Bioscope Catalyst, designed for fluid operation in contact or Tapping Mode.&#160; Also compatible with air operation.</td>
		</tr>
		<tr>
			<td>Cryoscopic osmometer; model OSMOMAT 030</td>
			<td>Gonotech, Berlin, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog Medium</td>
			<td>Duchess Biochemie</td>
			<td>M0221</td>
			<td>Original concentration, (1962)</td>
		</tr>
		<tr>
			<td>Optical inverted microscope coupled to the AFM</td>
			<td>Olympus IX81, Miami, FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PEGAMIL</td>
			<td>ANAEROBICOS S.R.L., Buenos Aires, Argentina</td>
			<td>100429</td>
			<td>Neutral, non acidic silicone glue</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Deltalab</td>
			<td>200201.B</td>
			<td>Polystyrene, 55 x 14 mm, radiation sterile.</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td>For root viability test.</td>
		</tr>
		<tr>
			<td>Silicon nitride probe, DNP-10, cantilever A</td>
			<td>Bruker, Billerica, MA, USA</td>
			<td>DNP-10/A</td>
			<td>For force modulation microscopy in liquid operation. Probe tip radius of 20-60 nm. 175-&#956;m-long triangular cantilever,&#160; with a spring constant of 0.35 N/m.</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Sigma</td>
			<td>T4537</td>
			<td></td>
		</tr>
	</tbody>
</table>

atomic force microscopy to study the physical properties of epidermal cells of live arabidopsis roots

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through nanoindentations with an atomic force microscope (AFM) coupled with an optical inverted fluorescence microscope. The method consists of applying controlled forces to the sample while measuring its deformation, allowing quantifying parameters such as the apparent Young's modulus of cell walls at subcellular resolutions. It requires a careful mechanical immobilization of the sample and correct selection of indenters and indentation depths. Although it can be used only in external tissues, this method allows characterizing mechanical changes in plant cell walls during development and enables the correlation of these microscopic changes with the growth of an entire organ.

Force-Indentation experiments 
The following text presents some results expected when a force-indentation experiment is conducted to show the typical output to expect when the protocol is well executed.
Force-displacement curves 
Representative force indentation plots that were obtained indenting live samples at a position placed in the center of the cell of the root elongation zone are presented in Figure 2. When the AF...

Watch this Scientific Journal Video about Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots at JoVE.com

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

The atomic force microscopy indentation protocol offers the possibility to dissect the role of the physical properties of the cell wall of a particular cell of a tissue or organ during normal or constrained growth (i.e., under water deficit).

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through ...

atomic-force-microscopy-to-study-physical-properties-epidermal-cells

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Developmental Biology

2.0K Views.  Universidad de la República (UdelaR). The atomic force microscopy indentation protocol offers the possibility to dissect the role of the physical properties of the cell wall of a particular cell of a tissue or organ during normal or constrained growth (i.e., under water deficit).

Video: Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in indentation mode, we show how to measure an elastic modulus and use it to evaluate cell wall mechanical properties. In addition, we also explain how to evaluate turgor pressure. The main advantages of atomic force microscopy are that it is non-invasive, relatively rapid (5~20 min), and that virtually any type of living plant tissue that is superficially flat can be analyzed without the need for treatment. The resolution can be very good, depending on the tip size and on the number of measurements per unit area. One limitation of this method is that it only gives direct access to the superficial cell layer.

Here, we present atomic force microscopy (AFM), operated as a nano- and micro-indentation tool on cells and tissues. The instrument allows the simultaneous acquisition of 3D surface topography of the sample and its mechanical properties, including cell wall Young's modulus as well as turgor pressure.

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in ...