This work was supported by grants from the Belgian National Fund for Scienti&#64257;c Research (FNRS), the Interuniversity Attraction Poles Programme-Belgian Science Policy and the &#8220;Communaut&#233; fran&#231;aise de Belgique-Actions de Recherches Concert&#233;es&#8221; to FC, from the Israel Science Foundation Jerusalem (ISF) to MM (Grant # 1311/12), and to NM (Grant # 1312/12).

1. Preparation of Solutions
<ol>
	<li>Prepare isotonic (600 mOsm) and hypotonic (500 mOsm) solutions containing 10 mM KCl, 1 mM CaCl2, and 8 M 2-(N-morpholine)-ethanesulphonic acid (MES), pH 5.7 and adjust osmolarity with the appropriate amounts of D-sorbitol: 540 mM for the isotonic and 440 mM for the hypotonic solution. Verify the osmolarity of the solution (within 3 % of the target value) using an osmometer.</li>
	<li>Prepare a dry stock of &#8216;enzymatic mix&#8217; containing the following enzymes: 0.55 g cellulase, 0.1 g pectolyase, 0.33 g polyvinylpyrrolidone K 30, 0.33 g BSA (see Table 1 below), mix the dry powder by vortex, make 5.7 mg aliquots and store at -20 &#176;C.</li>
</ol>
2. Isolation of Arabidopsis Mesophyll Protoplasts
<ol>
	<li>Prepare a Petri dish (10 cm) with about 6 drops (approx. 30 &#181;l each) of isotonic solution.</li>
	<li>Peel the abaxial (lower) Arabidopsis leaf epidermis, cut the peeled leaf into squares of about 4 x 4 mm2, then place the squares on the isotonic solution drops with the exposed abaxial side down, touching the solution.</li>
	<li>Dissolve 5.7 mg of the enzyme mix in 165 &#181;l isotonic solution (3.3 % w/w) in a 1.5 ml tube, mix gently by pipetting for a minute or so until dissolved, and place several similar drops of the enzymatic solution in the same Petri dish.</li>
	<li>Transfer the leaf pieces onto the enzymatic solution drops, close the dish sealing the lid with one round of parafilm and incubate for 20 min, floating the dish in a water bath set to 28 &#176;C.</li>
	<li>Add several more drops of the isotonic solution to the dish (2 drops per each enzyme solution drop). Transfer each leaf piece to a new isotonic solution drop, then, sequentially, to a second drop (to wash the enzymatic solution away). Lift the piece by its edge using forceps, shake it in the second drop (like a tea bag) to release the protoplasts. Collect the drops with the protoplasts (using a clipped off 100 &#181;l pipette tip) into a 1.5 ml tube.</li>
</ol>
3. The Hypotonic Challenge Assay: Arabidopsis Mesophyll Cell Swelling
<ol>
	<li>Prepare the perfusion system (Figure 1A) by filling one column with the isotonic solution and another column with the hypotonic solution. Open the valve, let some solution flow (first the hypotonic, then the isotonic) to fill the tubing all the way down to the inlet manifold (Figure 1B). Ensure there are no trapped air bubbles, and then close the valve.</li>
	<li>Seal a coverslip, using silicone grease (Table 1), to make a bottom for the chamber within the plexiglass slide (Figure 1B; see also the schematics of the chamber in Figure 1C). To make the chamber bottom (the upward facing exposed surface of the coverslip within the grease ring) &#8220;sticky&#8221; for protoplasts, coat it with positive-charge-bearing protamine sulphate (1 % in water; Table 1) or poly-L-Lysine (0.1 % in water; Table 1). Spread this &#8216;glue&#8217; over the coverslip using a pipette tip, wait for 1 - 2 min, rinse 3 - 4 times with the isotonic solution and shake away the remaining solution.</li>
	<li>Fill the chamber up with the isotonic solution. Then, add a drop of protoplasts containing solution to the chamber, using a clipped off pipette tip and wait 3 - 4 min for the protoplasts to settle. Cover the chamber with a transparent cover (Figures 1D, 1E) touching the solution surface (avoid trapping air bubbles beneath).</li>
	<li>Place the slide (gently!) on an inverted microscope table, connect it to the perfusion system and the pump (guarding against air bubbles in the tubing!) and turn on the isotonic solution flow for constant perfusion at 1 ml/min (faster rates can be used, up to 4 ml/min).</li>
	<li>For recording volume changes, an inverted microscope is used, with a 20X objective and with a CCD video camera connected to a PC computer. Use the &#8216;CMU 1394 Camera Driver&#8217; plugin of the ImageJ software (see the Table of Specific Materials for the download addresses of these two software pieces) to record a 60 sec video movie of selected immobile protoplasts (presumably, those stuck to the bottom) at a rate of 1 image /sec (1 Hz). Start the recording with a 15 sec wash of the isotonic solution (this constitutes the baseline), switch to the hypotonic solution for 45 sec (to complete a total 60 sec from the start of perfusion). Save the movie in TIF format. NOTE: Choose a view field with as many cells as possible, fulfilling the following criteria: spherical in shape and with a well-focused cell contour at their largest perimeter (Figure 2A).</li>
</ol>
4. Analysis of the Cell Volume Change Using ImageJ
NOTE: To analyze the series of images of a swelling cell, use the &#8216;Image Explorer&#8217; and &#8216;Protoplast Analyzer&#8217; plugins in the ImageJ software (written by Xavier Draye)14. Starting with the chosen protoplasts at their first time point, the &#8216;Protoplast Analyzer&#8217; plugin will detect automatically the protoplasts edges (contours) and calculate the time course of their areas during the experiment (the plugins are available with the PfFit analysis program, below).
<ol>
	<li>Start ImageJ. To open the movie, click &#8216;File&#8217; on the ImageJ panel, then, consecutively on the dropdown menus as they unfold: &#8216;Import&#8217; then &#8216;Image Explorer&#8217;. Highlight the chosen movie, then right-click on it, then left-click on &#8216;Protoplast Analyzer&#8217;. Browse through the movie (using a slider at the protoplast image bottom) to identify protoplasts that remain largely immobile during the experiment &#8211; these will be analyzed. Back on the first image, using the mouse, draw circles (picked from the ImageJ drawing tools) around the selected protoplasts (Figure 2B), then click &#8216;OK&#8217; in the table of &#8216;Detection parameters&#8217; that appeared.</li>
	<li>To launch the protoplast detection algorithm, click &#8216;Local&#8217; on the protoplast image top panel, then &#8216;Process&#8217; in the dropdown menu. Examine the green circles around the selected protoplasts (Figure 2C) throughout the movie. Save the &#8216;Result&#8217; in an Excel file. Quit ImageJ. NOTE: In case a red dot appears (to indicate a bad contour fit &#8211; usually due to a poor image contrast), rerun with different parameters.</li>
	<li>To separate the lines belonging to each cell (which &#8211; if two or more cells were analyzed simultaneously &#8211; will be intertwined, because the analysis is done frame by frame), in Excel, sort the saved data by the cell number column (&#8216;object&#8217;).</li>
	<li>To determine the pixel-to-&#181;m conversion factor for obtaining the real value of Pf, snap an image of a micrometer ruler via the same 20X microscope objective. Drag a line (picked from the ImageJ drawing tools) along the ruler image and read the pixel number equivalent to the ruler length at the bottom of the ImageJ main panel. Convert the arbitrary pixel area values in the Excel file into &#181;m2. Save the areas time course (for each cell separately) as a text file (two columns of numbers only). NOTE: This will be an input to the volume-fitting &#8216;PfFit&#8217; program.</li>
</ol>
5. Modeling the Rate of Osmolarity Change in the Experimental Chamber Using ImageJ and the Matlab Program PfFit
<ol>
	<li>Add 2 mg xylene cyanol (Table 1, below) to 100 ml of the isotonic solution (to produce the &#8216;Indicator Dye&#8217;).</li>
	<li>Prepare the perfusion system (as in 3.1) with the Indicator Dye and the non dyed hypotonic solution.</li>
	<li>Seal a cover slip using silicon grease to the bottom of the Plexiglass chamber, then gently fill the chamber with the Indicator Dye, cover it with a cover slip (as with the protoplasts before) and place it on the microscope stage.</li>
	<li>Connect the chamber to the perfusion system and the pump, and turn on the Indicator Dye flow for a constant perfusion at l ml/min.</li>
	<li>Record a 60 sec movie at the rate of 1 Hz. Start the recording with 15 sec of Indicator Dye, switch to the hypotonic solution for 45 sec. Stop filming. Flush with the Indicator Dye (at least for 30 sec), then start a new movie. Repeat about 5 - 6 times and save all the movies</li>
	<li>Use the ImageJ software to analyze the video images of the Indicator Dye transmittance to obtain an averaged time course of the changing transmittance.
	<ol>
		<li>Start ImageJ, click &#8216;File&#8217;, then, &#8216;Open&#8217;, and browse for the movie. For each movie, draw a 10 pixel wide vertical rectangle anywhere on the 1st image of the movie. Click &#8216;Image&#8217; on the ImageJ main panel, then click &#8216;Crop&#8217; in the dropdown menu.</li>
		<li>To align the 60 frames (of the 60 sec movie) in one row, click again &#8216;Image&#8217;, then click consecutively in the dropdown menus as they unfold: &#8216;Stacks&#8217; and &#8216;Make Montage&#8217; (columns 60, rows 1). Draw a 1 pixel high horizontal rectangle anywhere along the whole row of images and click &#8216;Analyze&#8217; in the ImageJ main panel, then click &#8216;plot profile&#8217; in the dropdown menu. NOTE: A &#8216;Plot of Montage&#8217; window will appear (not shown), and a list of transmittance data can be opened from its menu. Each image of the movie is represented in this list by 10 transmittance values originating in its 10 pixel wide rectangle and consequently the &#8220;time base&#8221; (the image sequential number) is 10 times longer.</li>
		<li>Copy the lists of the transmittance data (one list per movie) to an Excel file. Average the transmittance time courses obtained from the several movies of the Indicator Dye flushes. Generate a real time base by multiplying the image sequential number by 0.1. Save the averaged time course (two columns) to a text file. NOTE: Before averaging, if desired, plot the individual time courses, to reject any irregularities. Ensure that the movie includes at least 5 final sec of steady-state transmittance of the Indicator Dye.</li>
	</ol>
	</li>
	<li>Start the Matlab fitting program PfFit (the &#8216;Indicator Fit&#39; panel, Figure 3) to compute the various parameters of the osmolarity time course. NOTE: based on the known initial and final concentrations of the solution in the bath, the time course of the changing osmotic concentration of the solution is calculated from the concentration time course (calculated, in turn, from the Indicator Dye transmittance), assuming it follows the same dynamics as the dye concentration. PfFit is a program available for use free of charge. The &#8216; PfFit_Installer_web.exe&#8217; can be downloaded from: PfFit User Guide&#8217; with detailed explanations and definitions is accessible via Jove as a Supplemental file, which helps to familiarize the user with the PfFit program.</li>
	<li>In the &#8216;Indicator Fit&#39; panel, import the data of the mean time course of the Indicator Dye transmittance (&#8216;Indicator data file&#8217;, Figure 3A) and insert manually the current experiment parameters and the initial guesses of the parameters &#8216;width&#8217; and &#8216;t_half&#8217; describing the time course of the Indicator Dye concentration (Figure 3B. Click &#8216;Run&#8217; to view the plots of the time courses of the Indicator Dye concentration (real data and fit, Figure 4A), and of the modeled (calculated) bath osmolarity (Figure 4B). NOTE: a good fit to the data is essential(a recommendation: start with the values shown in Figure 3).</li>
</ol>
6. Determining the Pf using the Matlab Fitting Program PfFit
NOTE: In addition to the basic assumptions with regard to the behavior of a protoplast as a true and perfect osmometer11, the determination of Pf rests on the presumption that Pf may change with time, that this dynamics of Pf underlies the time course of the cell volume change and that three parameters suffice to describe it: Pfi (the initial value of Pf), SlopePf (the rate of the linear change of Pf) and Delay (the period from the start of the bath osmolarity change till the start of the cell volume change). Different models can be tested, including different combinations of these parameters and their values, including null values11. PfFit searches for the best combination of these parameters to yield &#8211; by calculation &#8211; the most faithful reproduction of the experimental time course of the cell volume change11, calculated, in turn, from the imported series of cell-contour areas (see also the Supplemental &#8216;PfFit User Guide&#8217;).
<ol>
	<li>Switch to the &#8216;Volume Fit&#8217; panel (Figure 5). Choose for import the areas data file (the text file with the time course of the &#8216;areas&#8217; of the analyzed protoplasts, Figure 5A). Choose &#8216;Last Indicator Fitting&#8217; as the parameter source (Figure 5B; see the &#8216;PfFit User Guide&#8217; for alternatives). NOTE: These parameters (Figure 5D) are then used to regenerate the osmoticum change in the bath for the volumes fitting procedure.</li>
	<li>In the &#8216;Volume Fit&#8217; panel (Figure 5C), initialize (fill in the initial guesses for) the Pf parameters: Pf, SlopePf and Delay (a recommendation: start with 1, 1, and 30, respectively), Chose the model &#8216;Class&#8217; (a recommendation: start with II and mark &#8216;checks&#8217; for all three parameters to be fitted). Click &#8216;Run&#8217;, then eyeball the interim figure (Figure 5E) and adjust the Delay parameter and the length of the record, if needed.</li>
	<li>Examine the results graph (Figure 6) to evaluate the fit quality and record the fit error. Change the initializing parameters a few fold each, and re-&#8216;Run&#8217;. NOTE: Don&#8217;t be discouraged when the program gets stuck &#8211; just restart the program!</li>
	<li>Repeat this procedure several times, starting with different combinations of initialization parameters, aiming for the lowest value of the fit error.</li>
	<li>Copy the list of the fit results directly from the screen, or find them in the PfFit-generated &#8216;_FIT_Vol_Results.txt&#8216; file.</li>
</ol>

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We have nothing to disclose.

Described here is a simple and very efficient procedure for measuring the Pf of isolated plant protoplasts, applicable in principle also to other spherical cells, e.g., frog oocytes11. This method is based on measuring the Pf in response to an osmotic challenge to the cell. In contrast to the other methods based on this approach, however, the change of solutions, i.e., of the osmolarity, is not instantaneous, but gradual, during a constant bath perfusion, starting with t...

Water uptake and flow across cellular membranes is a fundamental requirement for plant existence at both the cellular and the whole plant levels. At the cellular level, aquaporins (AQPs) play a key role in the regulation of the osmotic water permeability coefficient (Pf) of the cell membrane1-3.
To date, several methods have been employed in measuring the endogenous Pf of protoplast from different plant organs (i.e. roots, mesophyll, endodermis, etc., reviewed by Chaumont et al. 4). One of the approaches to measure Pf is to expose the protoplasts to an osmotic challenge and to monitor the initial rate of its volume change (i.e., the slope of the early linear phase of the volume change). Two different methods were previously described based on this approach, both based on an instantaneous exchange of solutions. The first one consists of immobilizing the protoplast with a suction micropipette and switching the solution flow5 and the second one of transferring the protoplast from one solution to another using a micropipette6. These micropipette suction and micropipette transferring methods, which allow image acquisition at the very start of the fast solution exchange (to capture the early linear phase of volume change), likely involve a physical stress to protoplasts and require specialized equipment and expert micromanipulation.
The method described here minimizes the disturbance to the cells, involves no micromanipulation and permits derivation of Pf when the bath perfusion is not instantaneous.
After the enzymatic digestion, the protoplasts, submerged in an isotonic solution, are immobilized on the coverslip-glass bottom of a Plexiglass (aka Lucite or perspex) chamber by charge interaction. Then, during a constant bath perfusion, the isotonic solution is flushed away by a hypotonic solution generating a hypoosmotic challenge to the protoplasts. The swelling of the protoplast is video recorded and then, by combining the information about the time course of the bath perfusion and the time course of the cell swelling, the Pf is determined by image processing and curve fitting procedures.
The advantages of this method are that the experiment is very efficient, i.e. it is possible to monitor a few cells simultaneously in a single assay, and that it does not require special equipment or particular micromanipulation skills. Several applications for this method are possible. For example, determination of the native Pf of a variety of cells from different tissues and plants, such as mesophyll and bundle sheath cells from Arabidopsis leaf7, maize leaf mesophyll or root cortex cells8-10 or suspension cultured cells11,12. In addition, it is possible to determine Pf of spherical animal cells such as oocyte cells11. Another example involves examination of AQP activity by transient expression of their gene in the protoplasts (or any other genes which may affect them; e.g., genes of kinases) and determination of their contribution to Pf; for example, expression of tomato AQP SlTIP2;2 in Arabidopsis mesophyll protoplasts by PEG transformation and determination the SlTIP2;2-related Pf13. Finally, examination of the effect on Pf of different molecules/substances (drugs, hormones, etc.) added to the solutions can also be examined, for example of the AQP blocker HgCl27.
The following protocol describes the isolation of protoplasts of Arabidopsis mesophyll cells and determination of their Pf.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="48">
			<td height="48" style="height:48px;width:288px;">KCl</td>
			<td style="width:140px;">Chem-Impex International</td>
			<td style="width:159px;">01247-1</td>
			<td style="width:516px;"><a href="http://www.chemimpex.com/">http://www.chemimpex.com</a> 
			Any source, anal. grade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:288px;">CaCl2</td>
			<td style="width:140px;">Merck</td>
			<td style="width:159px;">11718006</td>
			<td><a href="http://www.merck.com/">http://www.merck.com</a> 
			Any source, anal. grade</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:288px;">2-(N-morpholine)-ethanesulphonic acid (MES)</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">15152002</td>
			<td>http://www.sigmaaldrich.com 
			Any source, anal. grade</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:288px;">D-Sorbitol</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">18032003</td>
			<td>http://www.sigmaaldrich.com 
			Any source, anal. grade</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:288px;">Cellulase</td>
			<td style="width:140px;">Worthington, Lakewood, NJ, USA</td>
			<td style="width:159px;">LS002603</td>
			<td><a href="http://www.worthingtonbiochem.com/">http://www.worthingtonbiochem.com</a></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:288px;">Pectolyase</td>
			<td style="width:140px;">Karlan,&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Phonix, AZ, USA</td>
			<td style="width:159px;">8006</td>
			<td><a href="http://www.karlan.com/">http://www.karlan.com</a></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:288px;">Polyvinyl-pyrrolidone K 30 (PVP)</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">81420</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:288px;"><a href="https://my.labguru.com/catalog/materials/216">Bovine Serum Albumin (BSA)</a></td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">A9418-5G</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com</a></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:288px;">Protamine sulphate</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">P4380</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:288px;">Poly-L-Lysine</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">P8920</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:288px;">Xylene cyanol</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">X4126</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:288px;">Silicone vacuum grease heavy</td>
			<td style="width:140px;">Merck</td>
			<td style="width:159px;">107921</td>
			<td><a href="https://merck-chemicals.co.id/chemicals/silicone-high-vacuum-grease-heavy/MDA_CHEM-107921/p_LMib.s1Oxr4AAAEvXHg49in.?SecurePage=true&amp;SEO_ErrorPageOccurred=true&amp;attachments=CoA">https://merck-chemicals.co.id/chemicals/silicone-high-vacuum-grease-heavy/MDA_CHEM-107921/p_LMib.s1Oxr4AAAEvXHg49in.?SecurePage=true&#38;SEO_ErrorPageOccurred=true&#38;attachments=CoA</a></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Inverted microscope&#160;</td>
			<td>Nikon</td>
			<td>Eclipse TS100/TS100F</td>
			<td><a href="http://www.nikoninstruments.com/">http://www.nikoninstruments.com</a></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Peristaltic pump</td>
			<td>BIO-RAD</td>
			<td style="width:159px;">EP-1 Econo&#160;Pump</td>
			<td><a href="http://www.bio-rad.com/">http://www.bio-rad.com</a></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Grayscale digital camera</td>
			<td>Scion Corporation</td>
			<td style="width:159px;">CFW-1308M</td>
			<td><a href="http://www.scioncorp.com/">http://www.scioncorp.com</a></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">CMU 1394 Camera Driver&#8217; plugin for ImageJ</td>
			<td style="width:140px;">Carnegie Mellon</td>
			<td></td>
			<td><a href="http://www.cs.cmu.edu/~iwan/1394/download.html">http://www.cs.cmu.edu/~iwan/1394/download.html</a> 
			Free software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td style="width:140px;">NIH</td>
			<td></td>
			<td><a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</a> 
			Free software</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Econo Gradient Pump Fittings Kit</td>
			<td style="width:140px;">BIO-RAD</td>
			<td style="width:159px;">731-9006</td>
			<td><a href="http://www.bio-rad.com/">http://www.bio-rad.com</a></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Connectors, manifold</td>
			<td style="width:140px;">DirectMed</td>
			<td></td>
			<td><a href="http://directmed.com/main/Plastic-Medical-Tubing-Connectors.html?ACTION=S">http://directmed.com/main/Plastic-Medical-Tubing-Connectors.html?ACTION=S</a></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Burette infusion sets (columns)</td>
			<td style="width:140px;">Welford</td>
			<td style="width:159px;">IF-BR-001</td>
			<td>http://www.welfordmedical.com/content.php?id=61</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tubing</td>
			<td style="width:140px;">TYGON</td>
			<td style="width:159px;">R-3603</td>
			<td><a href="http://www.usplastic.com/">http://www.usplastic.com</a></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Plexiglass slide etc.</td>
			<td style="width:140px;">Perspectiv</td>
			<td></td>
			<td><a href="http://www.perspectiv.co.il/index-en.html">http://www.perspectiv.co.il/index-en.html</a> 
			Our slide was custom-made, it does not appear on the web site but a copy can be remade to order as &#39;a copy of the slide already made for M. Moshelion&#39;.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">3M packaging Scotch tape 1&#39;&#39;, clear</td>
			<td style="width:140px;">Viking Industrial, UK</td>
			<td style="width:159px;">VKMONO25</td>
			<td><a href="http://www.vikingtapes.co.uk/c-428-vkmono-mono-filament-tape.aspx#.UuvqOftdy_8">http://www.vikingtapes.co.uk/c-428-vkmono-mono-filament-tape.aspx#.UuvqOftdy_8</a> 
			any clear adhesive tape (sellotape, etc.) is likely to be OK</td>
		</tr>
	</tbody>
</table>

measuring the osmotic water permeability coefficient (pf) of spherical cells: isolated plant protoplasts as an example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

In order to determine the Pf and compare the activity of different AQPs, mesophyll protoplasts from Arabidopsis leaf are used. These protoplasts were found to have low basal (background) Pf levels 7 and can serve as a functional-expression system to enable reproducible Pf measurements.
Protoplasts from a mature leaf from a 6 week old Arabidopsis plant were isolated and three gene constructs with AQP genes from Arabidopsis (AtPIP2;1) and maiz...

Watch this Scientific Journal Video about Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example at JoVE.com

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

measuring-osmotic-water-permeability-coefficient-pf-spherical-cells

Research

JoVE Journal

Biology

15.9K Views.  The Hebrew University of Jerusalem. Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Video: Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network function1. These proteins are found at the presynaptic and postsynaptic membranes 2. Studies in mice indicate that neurexins and neurologins have an essential role in synaptic transmission 1. Recent reports have shown that altered neuronal connections during the development of the human nervous system could constitute the basis of the etiology of numerous cases of autism spectrum disorders 3.
Caenorhabditis elegans could be used as an experimental tool to facilitate the study of the functioning of synaptic components, because of its simplicity for laboratory experimentation, and given that its nervous system and synaptic wiring has been fully characterized. In C. elegans nrx-1 and nlg-1 genes are orthologous to human NRXN1 and NLGN1 genes which encode alpha-neurexin-1 and neuroligin-1 proteins, respectively. In humans and nematodes, the organization of neurexins and neuroligins is similar in respect to functional domains.
The head of the nematode contains the amphid, a sensory organ of the nematode, which mediates responses to different stimuli, including osmotic strength. The amphid is made of 12 sensory bipolar neurons with ciliated dendrites and one presynaptic terminal axon 4. Two of these neurons, named ASHR and ASHL are particularly important in osmotic sensory function, detecting water-soluble repellents with high osmotic strength 5. The dendrites of these two neurons lengthen to the tip of the mouth and the axons extend to the nerve ring, where they make synaptic connections with other neurons determining the behavioral response 6.
To evaluate the implications of neurexin and neuroligin in high osmotic strength avoidance, we show the different response of C. elegans mutants defective in nrx-1 and nlg-1 genes, using a method based on a 4M fructose ring 7. The behavioral phenotypes were confirmed using specific RNAi clones 8. In C. elegans, the dsRNA required to trigger RNAi can be administered by feeding 9. The delivery of dsRNA through food induces the RNAi interference of the gene of interest thus allowing the identification of genetic components and network pathways.

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are membrane-neuron adhesion proteins which perform essential roles in synaptic differentiation and transmission. Neuroligin deficient mutants of C. elegans are defective in detecting osmotic strength, but when they also contain a mutation in the gene coding neurexin, they recover the wild type phenotype.

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

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 ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. Furthermore, chloroplasts are critical for plant responses to various abiotic stresses, such as salinity and osmotic stresses. A chloroplast may contain up to ~3,000 different proteins, some of which are encoded by its own genome. However, the majority of chloroplast proteins are encoded in the nucleus and synthesized in the cytosol, and these proteins need to be imported into the chloroplast through translocons at the chloroplast envelope membranes. Recent studies have shown that the chloroplast protein import can be actively regulated by stress. To biochemically investigate such regulation of protein import under stress conditions, we developed the method described here as a quick and straightforward procedure that can easily be achieved in any laboratory. In this method, plants are grown under normal conditions and then exposed to stress conditions in liquid culture. Plant material is collected, and chloroplasts are then released by homogenization. The crude homogenate is separated by density gradient centrifugation, enabling isolation of the intact chloroplasts. Chloroplast yield is assessed by counting, and chloroplast intactness is checked under a microscope. For the protein import assays, purified chloroplasts are incubated with 35S radiolabeled in vitro translated precursor proteins, and time-course experiments are conducted to enable comparisons of import rates between genotypes under stress conditions. We present data generated using this method which show that the rate of protein import into chloroplasts from a regulatory mutant is specifically altered under osmotic stress conditions.

Here we describe a new method to study protein import into isolated chloroplasts under stress. The method is rapid and straightforward, and can be applied to study the consequences of different stress conditions for chloroplast protein import, and the corresponding regulatory mechanisms.

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. ...