This work was funded by grants to DMH and TDM (NHMRC project grants). DMH is a Grimwade Fellow, funded by the Miegunyah Trust.

1. Preparation of cells for labeling with ReAsH/FlAsH
<ol>
	<li>Using standard cell culture methods for your cell line of interest, prepare a culture of adherent cells directly in a live cell imaging slide ready for transfection.</li>
	<li>Transfect your plasmid containing TC-tagged gene of interest according to your transfection method of choice.</li>
</ol>
Note it is important to use positive and negative controls to assess the extent of specific binding to the TC tags and to assess for bleedthrough of fluorescence between the channels when collecting confocal micrographs. Hence, for two colors (eg FlAsH/Cherry or ReAsH/CFP or ReAsH/GFP combinations), ensure samples are prepared for single colors (eg Fluorescent protein alone or if possible a TC-tagged protein bound to FlAsH/ReAsH but with no fluorescent protein) 
<ol start="3">
	<li>One to two days after transfection, gently rinse the cells with 300 &mu;L pre-warmed (at 37 &deg;C) HBSS.</li>
	<li>Gently immerse the cells with 1 &mu;M FlAsH (or ReAsH) in 300 &mu;L of prewarmed HBSS and 10 &mu;M 1,2-ethanedithiol (EDT).</li>
</ol>
It is important to add EDT first before adding FlAsH/ReAsH and make the buffer just prior to adding it to the cells. Incubate for exactly 30 min at 37 &deg;C in tissue culture incubator. In our experience longer incubation times significantly increases the background fluorescence. New constructs should also be optimized for labeling time and FlAsH/ReAsH concentration (0.5-2 &mu;M).
<ol start="5">
	<li>Gently aspirate labeling solution from cells and then replace with 300 &mu;L prewarmed HBSS + 250 &mu;M 2,3-dimercaptopropanol (BAL) for 15 min at 37 &deg;C.</li>
	<li>Remove wash solution by gentle aspiration and replace with 300 &mu;L prewarmed HBSS.</li>
</ol>
After this wash, the cells may be fixed with paraformaldehyde (15 min with 3.2% solution), although we have found that this increases non-specific biarsenical dye fluorescence. Hence we usually image the cells live at room temperature. (Note that fixation of cells before labeling prevents biarsenical dye binding.)
2. Imaging the cells on a confocal microscope
<ol>
	<li>On the confocal microscope, set up parameters for imaging the individual fluorophores (see Table 1) and ensure that there is negligible bleedthrough 	between the channels. This can be achieved by checking individual fluorophores (in control samples) against each different fluorescent acquisition setting for fluorescence. Adjusting the emission wavelength range can help to minimize bleedthrough (although this can also reduce the signal/noise).</li>
	<li>Also adjust the photomultiplier settings so that the maximum fluorescence in the sample does not saturate the detector. (This can be detected using the Q-LUT setting on the SP2 Leica confocal). Once the best imaging settings are determined, do not change any of them between samples.</li>
	<li>Other settings we typically use (although these can be optimized) are a scan rate of 200 Hz and collect 4 line averages and a pinhole diameter of 1 Airy unit. The pinhole diameter can be expanded if signal/noise is an issue, however, this may result in some loss of imaging.</li>
	<li>Collect the confocal images in 12-bit (or higher) format if possible. 12-bit format captures a greater dynamic range of values (0-4095) for each pixel intensity than 8-bit (0-255). This is important for ensuring the richest data set is recorded, which maximizes quality of quantitative data analysis.</li>
	<li>Collect images for the fluorescent protein channel (Cherry, GFP or CFP) and also for the biarsenical dye channel (FlAsH or ReAsH) for all samples.</li>
</ol>
3. Analysis of the data
<ol>
	<li>Install the software ImageJ on your computer8. <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a></li>
	<li>Make sure that your version of ImageJ has the following plugins:
		<ul>
			<li><a href="http://rsbweb.nih.gov/ij/plugins/file-opener.html">http://rsbweb.nih.gov/ij/plugins/file-opener.html</a></li>
			<li><a href="http://rsbweb.nih.gov/ij/plugins/image_correlator.html">http://rsbweb.nih.gov/ij/plugins/image_correlator.html</a></li>
			<li><a href="http://rsbweb.nih.gov/ij/plugins/lut-editor.html">http://rsbweb.nih.gov/ij/plugins/lut-editor.html</a></li>
		</ul>
	</li>
	<li>Open ImageJ, and if using a version older than v1.32c, click on the following options to enable multiple images to be opened at one time (which can be done by holding down the control button while you click on different files): 
	<img src="/files/ftp_upload/2857/2857step1.jpg" alt="Figure 1" /></li>
	<li>Open the images of interest in ImageJ. 
	ImageJ will automatically define the maximum and minimum pixel intensities that are displayed on the screen for each image separately. Given that different images will have different pixel intensities, this means the displayed images ARE NOT comparable as viewed.</li>
	<li>To ensure the opened images are all on the same scale, you can physically define the upper and lower pixel intensities to be viewed (this will not change the actual data content of the images as would be the case in some other software). These values can be set under the following menu: 
	<img src="/files/ftp_upload/2857/2857step2.jpg" alt="Figure 2" /></li>
	<li>An alternative approach is to work with stacks, which puts all images together into one file and will automatically scale (for viewing) all the images in the stack to the same scale. The easiest way to work with the data is to convert each channel to a stack. Thus, for the fluorescent protein channel convert all to stack as shown. You can easily select one channel by stacking all images containing a common name (eg "ch00") in the title. 
	<img src="/files/ftp_upload/2857/2857step3.jpg" alt="Figure 3" /></li>
	<li>Now convert all the open images to 8-bit for analysis. This will actually rescale the viewed range into a 0-255 range (which defines 8-bit). 
	IMPORTANT: Do not save over the original 12-bit (or higher) format or you will lose information content. Note that some software packages can only display 8-bit images so this format is useful for making figures etc. 
	<img src="/files/ftp_upload/2857/2857step4.jpg" alt="Figure 4" /></li>
	<li>Save a copy in a new folder called "8-bit converted" using the "Save as&#x2026;" option.</li>
	<li>One method to examine the extent of biarsenical dye binding in a whole image is to perform a pixel intensity correlation plot. This plots every pixel value in one channel relative to the corresponding pixel value in a second channel. Hence a pixel position high in CFP fluorescence will also be high in ReAsH fluorescence if there is high binding.</li>
	<li>To analyze the pixel co-correlation, make sure the 8-bit ReAsH and Cerulean/GFP images are open in ImageJ.</li>
	<li>Open the "Image Correlator" plugin as indicated below. Select "Image1" as the ReAsH or FlAsH stack and "Image2" as the fluorescent protein stack. 
	<img src="/files/ftp_upload/2857/2857step5.jpg" alt="Figure 5" /></li>
	<li>The resulting stack may not display any detailed information - this is normal. Save the stack in a new folder called "Scatterplots" and give it the same file name as the sample it refers to (eg "FlAsH-cherry correlation plot")</li>
	<li>To view the data meaningfully you can do one of two options. First transform the data so that it is in log format. Then visually rescale the data as follows: 
	<img src="/files/ftp_upload/2857/2857step6.jpg" alt="Figure 6" /></li>
	<li>Alternatively, you can rescale the data visually to only show low values (eg 1-255) and display the data using a special LUT. An LUT (Look Up Table) represents a table of colors that are assigned to each pixel value in an image. This can be used to define a pseudocolor scheme for an image 	and is useful for defining certain features in an image. To create a custom LUT click on "LUT editor" and make a new one such as the one shown (this can be saved and used later on). 
	<img src="/files/ftp_upload/2857/2857step7.jpg" alt="Figure 7" /></li>
	<li>Set the brightness/contrast range from 0-255 (as described in step 13 above). To "lock in" the image as to that shown on the screen, the image can be converted to RGB format which saves an 8-bit value for each of the red, green and blue color of each pixel. 
	<img src="/files/ftp_upload/2857/2857step8.jpg" alt="Figure 8" /> 
	To copy and paste images to other programs first open the 8-bit or 16-bit images. As described in step 14 above, assign an LUT color scheme to the image. For CFP, assign the "Cyan" LUT as described below&#x2026; 
	<img src="/files/ftp_upload/2857/2857step9.jpg" alt="Figure 9" /></li>
	<li>Select the image and to copy it to the clipboard, select&#x2026; 
	<img src="/files/ftp_upload/2857/2857step10.jpg" /></li>
</ol>
4. Representative results:
The success of labeling cells with biarsenical dyes is dependent on a few key parameters. First, the timing of the labeling with 
dyes is crucial. We have found that extended periods of labeling (more than 30 min) results in a high level of non-specific background staining. Fig 1 shows a typical result for a wild-type form of huntingtin fragment (25Q) fused to the CFP derivative Cerulean containing a TC tag as described previously 7. This sample was stained for 30 min with ReAsH and there is minimal background in the sample lacking the TC tag. We have found that fixing cells with paraformaldehyde increases background while fixing with methanol abrogates the fluorescence of the fluorescent protein tag. Hence where possible we image the cells live. It is important to also note that fixation prior to labeling with biarsenical dyes prevents their binding, presumably due to modifications of the TC motif.
Another critical factor for consistent results is the density of cells. We have found it critical to image cells that are loosely distributed and also that extensive clumping can lead to uneven staining of the biarsenical dyes in different cells.
<img src="/files/ftp_upload/2857/2857fig1.jpg" alt="Figure 1" /> 
Figure 1. Tetracysteine tags and ReAsH staining in live cells transfected with huntingtin(exon1-25Q)-Cerulean fusions. The TC tag is located at the junction of the huntingtin-Cerulean fusion (as described in 7). The pixel intensity correlation plot enables an assessment to differences in ReAsH binding throughout the cell and can be used to map changes in ReAsH binding due to conformational change or ligand interactions.

<ol>
	<li>Tsien, R. 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+green+fluorescent+protein.">The green fluorescent protein.</a> Ann. Rev. Biochem. 67, 509-544 (1998).</li><li>Griffin, B. A., Adams, S. R., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+covalent+labeling+of+recombinant+protein+molecules+inside+live+cells.">Specific covalent labeling of recombinant protein molecules inside live cells.</a> Science. 281, 269-2672 (1998).</li><li>Uttamapinant, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorophore+ligase+for+site-specific+protein+labeling+inside+living+cells.">A fluorophore ligase for site-specific protein labeling inside living cells.</a> Proc. Natl. Acad. Sci. USA. 107, 10914-10919 (2010).</li><li>Ignatova, Z., Gierasch, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+protein+stability+and+aggregation+in+vivo+by+real-time+fluorescent+labeling.">Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling.</a> Proc. Natl. Acad. Sci. USA. 101, 523-528 (2004).</li><li>Coleman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+detection+of+prion+protein+with+biarsenical+labeling+and+FlAsH+fluorescence.">Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence.</a> Biochem. Biophys. Res. Commun. 380, 564-568 (2009).</li><li>Luedtke, N. W., Dexter, R. J., Fried, D. B., Schepartz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveying+polypeptide+and+protein+domain+conformation+and+association+with+FlASH+and+ReAsH.">Surveying polypeptide and protein domain conformation and association with FlASH and ReAsH.</a> Nat. Chem. Biol. 3, 779-784 (2007).</li><li>Ramdzan, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformation+sensors+that+distinguish+monomeric+proteins+from+oligomers+in+live+cells.">Conformation sensors that distinguish monomeric proteins from oligomers in live cells.</a> Chem. Biol. 17, 371-379 (2010).</li><li>Abramoff, M. a. g. e. l. h. a. e. s., PJ, S. J. R. a. m. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+with+ImageJ.">Image processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Hearps, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biarsenical+dye+Lumio+exhibits+a+reduced+ability+to+specifically+detect+tetracysteine-containing+proteins+within+live+cells.">The biarsenical dye Lumio exhibits a reduced ability to specifically detect tetracysteine-containing proteins within live cells.</a> J. Fluor. 17, 593-597 (2007).</li><li>Adams, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+biarsenical+ligands+and+tetracysteine+motifs+for+protein+labeling+in+vitro+and+in+vivo:+Synthesis+and+biological+applications.">New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications.</a> J. Am. Chem. Soc. 124, 6063-6076 (2002).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat. Meth. 2, 905-909 (2005).</li></ol>

No conflicts of interest declared.

The approach to label protein localization with a fluorescent protein and conformational properties with a second dye offers much 
potential for mapping where different conformations of proteins accrue in cells and events that change the dynamics of protein conformation. 
ReAsH/FlAsH was first used as an in-cell sensor for protein folding of the mammalian cellular retinoic acid-binding protein I 4. In this example, 
the FlAsH bound to a TC tag engineered into cellular retinoic acid-binding protein I had redu...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the Reagent</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>8-well &mu;-slides</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>We find these chamber slides to be particularly useful for culturing cells for imaging.</td>
		</tr>
		<tr>
			<td>TC-FlAsH II In-cell Tetracysteine Tag Detection Kit *green fluorescence* *for live-cell imaging</td>
			<td>Invitrogen</td>
			<td>T34561 (FlAsH) or T34562 (ReAsH)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hanks' Balanced Salt Solution </td>
			<td>Invitrogen</td>
			<td>14175-103</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2,3-Dimercapto-1-propanol</td>
			<td>Sigma-Aldrich</td>
			<td>D1129-5ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1,2-Ethanedithiol</td>
			<td>Sigma-Aldrich</td>
			<td>02390-25ML</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

reash/flash labeling and image analysis of tetracysteine sensor proteins in cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

Watch this Scientific Journal Video about ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells at JoVE.com

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

reashflash-labeling-image-analysis-tetracysteine-sensor-proteins

Research

JoVE Journal

Biology

16.9K Views.  Bio21 Molecular Science and Biotechnology Institute. The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

Video: ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...