Work in the authors' laboratory is supported by a grant from the National Institute of General Medical Sciences (GM41628) and by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.

1. ATP-dependent Nucleosome Remodeling Assays
To measure ATP-dependent nucleosome remodeling activities, immunopurified INO80 or INO80 subcomplexes are incubated with ATP and a mononucleosomal substrate, which contains a single nucleosome positioned at one end of a 216-bp, 32P-labeled DNA fragment. The reaction products are then subjected to electrophoresis in native poly-acrylamide gels.
<ol>
	<li>To generate the 32P-labeled, &#39;601&#39; DNA fragment, amplify from pGEM-3Z-6014 a 216 bp DNA fragment containing an end-positioned 601 nucleosome positioning sequence, using the oligonucleotides 5&#8242;-ACAGGATGTATATATCTGACACGTGCCTGG and 5&#8242;-AATACTCAAGCTTGGATGCCTGCAG as forward and reverse primers.
	<ol>
		<li>Set up a 100 &#956;l PCR reaction as described in Table 1.</li>
		<li>Perform the PCR reactions in a thermal cycler using the following program: 1 min at 96 &#176;C, followed by 45 sec at 94 &#176;C, 30 sec at 57 &#176;C, 60 sec at 72 &#176;C for 30 cycles; ending with 7 min at 72 &#176;C.</li>
		<li>After the PCR reaction is finished, remove the unincorporated nucleotides by passing the reaction products twice through nuclease-free spin columns.</li>
		<li>Run 5 &#956;l of the purified PCR product in a 1.2% agarose gel to confirm that the PCR reaction generated the desired ~216 bp DNA fragment.</li>
		<li>Dilute 5 &#956;l of the purified PCR product 20-fold, and measure the DNA concentration using a UV spectrophotometer. The average yield is ~40 ng/&#956;l.</li>
		<li>Measure the radioactivity of 1 &#956;l of the purified PCR product in a scintillation counter. Estimate the labeling efficiency by calculating the cpm/ng. A successful labeling reaction is expected to yield ~15,000 cpm/ng of 601 DNA fragment.</li>
	</ol>
	</li>
	<li>Transfer Hela cell nucleosomes onto the labeled 601 DNA using a serial dilution method 5.
	<ol>
		<li>Mix 2 pmol of 32P-labeled 601 DNA fragment with 6 &#181;g of Hela nucleosomes (prepared as described 5) in 50 &#181;l of a buffer containing 1.0 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1 mM PMSF, and 1 mM DTT and incubate at 30 &#176;C for 30 min.</li>
		<li>Sequentially dilute the mixture to 0.8 M, 0.6 M, and 0.4 M NaCl by dilution with 12.5 &#181;l, 20.8 &#181;l, and 41.6 &#181;l, respectively, of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1 mM PMSF, and 1 mM DTT, with a 30 min incubation at 30 &#176;C after each dilution.</li>
		<li>Further dilute the mixture to 0.2 M and then to 0.1 M NaCl by addition of 125 &#181;l and then 250 &#181;l of the same buffer containing 0.1% Nonidet P-40, 20% glycerol, and 200 &#181;g/ml BSA, with a 30 min incubation at 30 &#176;C between the final two dilutions. Store the mononucleosome substrate at 4 &#176;C for up to 3 months.</li>
	</ol>
	</li>
	<li>Perform ATP-dependent nucleosome sliding reactions in a total volume of 10 &#181;l. The reaction components are listed in Table 2. Since the optimal NaCl concentration for INO80 nucleosome remodeling activity is ~50 mM NaCl (unpublished results), adjust the total concentration of NaCl in each reaction to 50 mM, taking into account the amount of NaCl contained in preparations of nucleosomes and enzyme.
	<ol>
		<li>Before beginning to set up the assays, cast native poly-acrylamide gels (18 x 16 cm). To prepare a single gel containing 5% acrylamide (acrylamide:bisacrylamide 37.5:1), 0.5x TBE (45 mM Tris borate, 1 mM EDTA), 0.01% ammonium persulfate (APS), and 0.001% N,N,N&#180;,N&#180;-tetramethylethylenediamine (TEMED), mix the ingredients listed in Table 3. Allow the gel to polymerize for at least 2 hr at RT.</li>
		<li>Meanwhile, for each reaction to be performed, combine in a pre-chilled siliconized 1.5 ml microcentrifuge tube ~20 nM INO80 or INO80 subcomplex (prepared as described 3) with an amount of EB100 buffer (Table 4) sufficient to give a volume of 4.75 &#956;l. Immediately re-freeze any remaining INO80-containing fractions in powdered dry ice or liquid nitrogen.</li>
		<li>Set up a master cocktail with the rest of the ingredients, scaling up by a factor of &#39;X&#39; (where X = total number of reactions +3). The amount of each ingredient needed for a single 10 &#956;l reaction is listed in Table 5; the recipe should be scaled up according to the number of reactions to be performed. Mix well by tapping the tube or by pipetting up and down with a Pipetman and spin the tube for a few seconds in a benchtop microcentrifuge.</li>
		<li>Dispense 5.25 &#181;l of the master cocktail to each of the reaction tubes set up in step 1.3.2. Mix well by pipetting up and down. Start the reactions by transferring reaction tubes to a 30 &#176;C heat block or water bath and incubate for 2 hr.</li>
		<li>Meanwhile, prepare &#39;removing mix&#39; cocktail containing competitor DNA and nucleosomes, scaling up by a factor of X (X = total number of reactions + 4). The amount of each ingredient needed to prepare 1.5 &#181;l removing mix for a single reaction is listed in Table 6; the recipe should be scaled up depending on the number of assays to be performed.</li>
		<li>Terminate reactions by adding 1.5 &#956;l of the removing mix. Mix well, spin down, and incubate at 30 &#176;C for a further 30 min.</li>
		<li>Meanwhile, pre-run the native polyacrylamide gel in a vertical electrophoresis unit at 100 V for 30 min at 4 &#176;C, using 0.5x TBE as running buffer with a magnetic stir bar inside the lower chamber to maintain constant buffer circulation.</li>
		<li>To load the sample, add 2.5 &#181;l of loading dye containing 3x TBE, 30% glycerol, 0.25% Bromophenol Blue, and 0.25% Xylene Cyanol FF. Mix well, briefly spin the samples, and load onto the gel using loading tips.</li>
		<li>Run the gel at 200 V for 4.5 hr at 4 &#176;C with buffer circulation.</li>
		<li>To detect the signal, transfer the gel to a stack of two sheets of filter paper. Wrap the filter paper with the gel on top using clear plastic wrap, and then expose it to a storage phosphor screen at 4 &#176;C for the desired time.</li>
		<li>Scan the screen with an isotope imaging scanner system and analyze the data using suitable software.</li>
	</ol>
	</li>
</ol>
2. Mononucleosome Binding Assays
To assay the binding affinity of a given INO80 complex for mononucleosomes, perform an Electrophoretic Mobility Shift Assay (EMSA) using the mononucleosomal substrate generated in Step 1.2.
<ol>
	<li>Set up the reaction mixes for binding assays as described for nucleosome remodeling assays but omit the ATP and removing mix from the reactions; incubate at 30 &#176;C for 30 min.</li>
	<li>Add 2.5 &#181;l of loading dye to each reaction mixture, and apply to a native polyacrylamide gel containing 3.5% acrylamide (acrylamide:bis 37.5:1), 1% Glycerol, 0.5x TBE, 0.01% APS, and 0.001% TEMED.</li>
	<li>Using 0.5x TBE as running buffer, run the gel at 200 V for 2.5 hr at 4 &#176;C with buffer circulation and expose to a storage phosphor screen.</li>
</ol>
3. DNA- and Nucleosome-dependent ATPase Assays
Perform ATPase assays in 5 &#956;l reaction mixtures containing 20 mM Tris-HCl (pH 7.5), 60 mM NaCl, 6.6 mM MgCl2, 0.8 mM EDTA, 0.015% Nonidet P-40, 2.5% glycerol, 0.1 mg/ml BSA, 1 mM DTT, 0.1 mM PMSF, 2 mM ATP, 2 &#956;Ci of [&#945;-32P] ATP (3,000 Ci/mmol). For each INO80 complex or amount of INO80 complex to be assayed set up three parallel reactions, one containing EB100 buffer to measure DNA- or nucleosome-independent ATPase, one containing closed circular plasmid DNA (5,000 bp, ~30 nM) to measure DNA-dependent ATPase, and one containing Hela oligonucleosomes (~185 nM) to measure nucleosome-dependent ATPase. Set up all reactions on ice.
<ol>
	<li>For each reaction, combine 10-50 nM of the immunopurified INO80 or INO80 subcomplexes with an amount of EB100 buffer sufficient to give a volume of 2.2 &#956;l in pre-chilled lubricated 1.5 ml microcentrifuge tubes. Immediately re-freeze any INO80-containing fractions in powdered dry ice or liquid nitrogen.</li>
	<li>Set up a master cocktail. The amount of each ingredient needed for a single reaction is listed in Table 7. Scale up the recipe by scaling up by a factor of 3(X+2) +1, where X = the number of INO80 preparations to be assayed.</li>
	<li>To prepare &#39;sub-cocktails&#39; containing buffer only, DNA, or nucleosomes, dispense 2.5(X+2) &#956;l of the master cocktail into three separate tubes. Add 0.3(X+2) &#956;l of either EB100, closed circular plasmid DNA (1.5 &#181;g/&#181;l), or Hela oligonucleosomes (1.5 &#956;g/&#956;l) and mix well.</li>
	<li>Dispense 2.8 &#956;l of the appropriate sub-cocktail to the enzyme-containing reaction tubes set up in step 3.1. Gently pipette up and down to mix; avoid introducing bubbles.</li>
	<li>To start reactions, transfer the reaction tubes to a 30 &#176;C heat block.</li>
	<li>After 5, 15, 30, and 60 min of incubation, spot 0.5 &#956;l of each reaction mixture onto a cellulose polyethyleneimine thin layer chromatography (TLC) plate (20 x 10 cm) in a straight line at least 1.5 cm away from the bottom edge. Immediately return reaction tubes to the 30 &#176;C heat block so multiple time points can be taken from a single tube. After spotting, dry the TLC plates using a blow dryer.</li>
	<li>Transfer the TLC plates to a glass chamber containing enough 0.375 M potassium phosphate (pH 3.5) to allow the bottom 0.5 cm of the TLC plate to be submerged in the solution.</li>
	<li>Cover the chamber, and develop until the front of the liquid phase reaches the top of the TLC plates. Immediate dry the plates thoroughly using a blow dryer.</li>
	<li>Expose the dried TLC plates to a storage Phosphor screen at RT. Scan the screen with an isotope imaging scanner system and determine the amount of radioactive ATP substrate and ADP product.</li>
	<li>To calculate the amount of ATP hydrolyzed, multiply the % ATP hydrolyzed by the amount of ATP present in the starting reaction mixture using the following formula: pmol ATP hydrolyzed = 10 pmol ATP in starting reaction x [ADP/(ATP+ADP)]</li>
</ol>

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The authors declare that they have no competing financial interests.

To ensure that nucleosome remodeling and ATPase activities we observe in assays depend on the catalytic activity of INO80 complexes, and not on contaminating remodeling and/or ATPase enzymes, we routinely assay nucleosome remodeling and ATPase activity of catalytically inactive versions of INO80 complexes, purified in parallel with wild type INO80 using the same procedure. A negative control reaction lacking ATP should also be performed when assaying nucleosome remodeling activity to test for the presence of contaminatin...

SNF2 family chromatin remodeling complexes include a central SNF2-like ATPase subunit 1,2. Some SNF2-like ATPases function as single subunit enzymes, while others function as the catalytic subunit of larger multi-subunit complexes. Elucidating the molecular mechanisms by which each of the subunits of chromatin remodeling complexes contribute to their activities requires the ability to perform biochemical assays that dissect the remodeling process.
ATP-dependent nucleosome remodeling by the human INO80 complex and other chromatin remodeling enzymes can be envisioned as a multi-step process that starts with binding of the remodeling enzyme to nucleosomes, followed by activation of its DNA- and/or nucleosome-dependent ATPase, translocation of the remodeling enzyme on nucleosomal DNA, and eventual repositioning of nucleosomes 1,2. Understanding the molecular details of the ATP-dependent chromatin remodeling process requires dissection of the remodeling reaction into its individual steps and definition of the contributions of individual subunits of the chromatin remodeling complex to each step of the reaction. Such analyses require the ability to analyze nucleosome remodeling and other activities using defined molecular substrates in vitro.
In a previous JOVE protocol, we described procedures used to generate INO80 chromatin remodeling complexes and subcomplexes with defined subunit compositions 3. Here, we present three biochemical assays that enable quantitative analysis of the nucleosome binding, DNA- and nucleosome-activated ATPase, and nucleosome remodeling activities associated with such complexes.

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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Name of Reagent/Material</td>
			<td style="width:212px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:123px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">P8340</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x PCR reaction buffer&#160;</td>
			<td style="width:212px;">Roche Applied Science&#160;</td>
			<td style="width:147px;">11435094001</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roche Taq DNA Polymerase</td>
			<td style="width:212px;">Roche Applied Science&#160;</td>
			<td style="width:147px;">11435094001</td>
			<td style="width:123px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">NucAway Nuclease-free Spin Columns&#160;</td>
			<td>Ambion</td>
			<td style="width:147px;">Cat. # AM10070</td>
			<td style="width:123px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">ultrapure ATP&#160;</td>
			<td>USB/Affymetrix</td>
			<td style="width:147px;">77241 25 UM</td>
			<td style="width:123px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">bovine serum albumin&#160;</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">A9418&#160;</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">N,N,N&#180;,N&#180;-tetramethylethylenediamine (TEMED)</td>
			<td style="width:212px;">Thermo Scientific</td>
			<td style="width:147px;">17919</td>
			<td style="width:123px;">Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40% Acrylamide/Bis 37.5:1</td>
			<td style="width:212px;">Amresco</td>
			<td style="width:147px;">0254-500ML</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicated salmon sperm DNAs&#160;</td>
			<td>GE Healthcare</td>
			<td style="width:147px;">27-4565-01</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% ammonium persulfate (APS)</td>
			<td style="width:212px;">Thermo Scientific</td>
			<td style="width:147px;">17874</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">benzonase&#160;</td>
			<td>Novagen</td>
			<td style="width:147px;">Cat. No. 70664</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">[&#945;-32P] ATP (3000 Ci/mmol)</td>
			<td style="width:212px;">PerkinElmer</td>
			<td style="width:147px;">BLU003H250UC</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">dCTP, [&#945;-32P]- 6000Ci/mmol</td>
			<td style="width:212px;">PerkinElmer</td>
			<td style="width:147px;">BLU013Z250UC</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;"></td>
			<td style="width:212px;"></td>
			<td style="width:147px;"></td>
			<td style="width:123px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Equipment</td>
			<td style="width:212px;">Company</td>
			<td style="width:147px;"></td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR thermal cycler PTC 200</td>
			<td>MJ Research</td>
			<td style="width:147px;">PTC 200</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoefer vertical electrophoresis unit</td>
			<td style="width:212px;">Hoefer</td>
			<td style="width:147px;">SE600X-15-1.5</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lubricated 1.5ml microcentrifuge tubes&#160;</td>
			<td style="width:212px;">Costar</td>
			<td style="width:147px;">3207</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Storage Phosphor Screen&#160;</td>
			<td>Molecular Dynamics</td>
			<td style="width:147px;">63-0034-79</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3MM filter paper</td>
			<td>Whatman&#160;</td>
			<td style="width:147px;">28458-005</td>
			<td style="width:123px;">VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Typhoon PhosphorImager&#160;</td>
			<td>GE Healthcare</td>
			<td style="width:147px;">8600</td>
			<td style="width:123px;"></td>
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			<td height="21" style="height:21px;">ImageQuant software</td>
			<td>GE Healthcare</td>
			<td style="width:147px;">ver2003.02</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">TLC Glass Plates, PEI-Cellulose F</td>
			<td style="width:212px;">Millipore</td>
			<td style="width:147px;">5725-7</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Immobilon-FL Transfer Membrane 7 x 8.4</td>
			<td style="width:212px;">Millipore</td>
			<td style="width:147px;">IPFL07810</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">General purpose survey meter with end-window or pancake GM (Geiger-Mueller) probe</td>
			<td style="width:212px;">Biodex</td>
			<td style="width:147px;">Model 14C</td>
			<td style="width:123px;"></td>
		</tr>
	</tbody>
</table>

biochemical assays for analyzing activities of atp-dependent chromatin remodeling enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

The figures show representative results of biochemical assays used to characterize INO80 activities, including nucleosome sliding (Figure 1) and binding (Figure 2) assays and DNA- or nucleosome-dependent ATPase assays (Figure 3).
The experiment shown in Figure 1 compares the ability of intact INO80 complexes purified through FLAG-Ies2 or FLAG-INO80E and of INO80 subcomplexes purified through either FLAG-Ino80&#916;N or Ino80&#...

Watch this Scientific Journal Video about Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes at JoVE.com

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

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14.7K Views.  Stowers Institute for Medical Research. Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Video: Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...