We thank members of the Meshorer lab, especially Shai Melcer, Adi Alajem, Edupuganti Raghu Ram, Badi Sri Sailaja, Anna Mattout and Alva Biran, for critical comments and for trouble-shooting photobleaching experiments on a daily basis. EM is a Joseph H. and Belle R. Braun Senior Lecturer in Life Sciences and is supported by the Israel Science Foundation (ISF 943/09), the Israel Ministry of Health (6007) the European Union (IRG-206872 and 238176), the Israel Cancer Research Foundation, the Internal Applicative Medical Grants of the Hebrew University and the Israel Psychobiology Institute.

1. Plating the ES Cells
T = 0 hrs 
MEF plating
<ol>
 <li>Coat the live imaging 8-well &mu;-Slides (ibidi; Munich, Germany) with gelatin or in chambered cover glasses (Lab-Tek; Rochester, NY) or in glass-bottom culture dishes (MatTek; Ashland, MA). Leave for 5-30 min and aspirate away the free gelatin.</li>
 <li>Seed 22,000 MEFs/well in 250 &mu;l total volume of DMEM [supplemented with 10% fetal bovine serum (FBS)]. Allow cells to grow in a tissue culture incubator (37&deg;C, 5% CO2).</li>
 </ol>
T = 6 hrs 
 ES cell plating
 <ol start="3">
 <li>Aspirate DMEM.</li>
 <li>Seed each MEF coated well with 15,000 R1 cells/well in 250 &mu;l total volume of ES cell media [supplemented with 10% ESC-grade fetal bovine serum (FBS), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.1 mM &beta;-mercaptoethanol, and 1000 U/ml leukemia inhibitory factor (LIF)], to obtain 30% to 50% confluence the following day.</li>
</ol>
2. Transfecting the ES Cells
T = 24 hrs 
Transient transfection
<ol>
 <li>Replace the ES cell media with 250 &mu;l/well of fresh ES cell media.</li>
 <li>In a 1.5-ml sterile test tube, add 100 &mu;l serum free media [Opti-MEM (Gibco)], then add 10 &mu;l TransIT-LT1 transfection reagent (Mirus) directly into the serum free media. Mix by gentle pipetting and incubate at room temperature for 5-20 min.</li>
 <li>Add 1.5 &mu;g GFP fusion plasmid DNA (H1e, H1o or HP1) to the diluted transit-LT1 reagent. Mix by gentle pipetting and incubate at room temperature for 15-30 min.</li>
 <li>Add 13.5 &mu;l/well of the transfection mixture. Swirl the 8-well &mu;-Slides to ensure even dispersal. After 24 hrs replace the old ES cell media with 250 &mu;l of fresh ES cell media.</li>
</ol>
3. Performing FRAP and FLIP
T = 48-72 hrs
<ol>
 <li>The experiment can be performed on any confocal laser scanning microscope (CLSM) but since in a normal FRAP/FLIP experiment, many consecutive images are acquired, it is recommended to use a spinning-disk confocal microscope, which enables acquisition speed and ensures that no undesired sample bleaching will occur following the initial intentional bleaching event. Here, we recommend using the Revolution spinning disk confocal system (www.Andor.com), with the Yokogawa CSU-X spinning disk head. This system has the dual capacity to photobleach using a specialized FRAPPA module with a point scanning system, and to rapidly switch the light back to collect images using the spinning disk. The 3 most common fluorescing proteins used for photobleaching experiments are GFP, YFP and Cherry. If GFP or YFP are used, a ~488 nm laser is required. For Cherry, use a ~560 nm laser. In all cases, we recommend using solid state lasers. Having an automated stage is useful but not required. Since live cells are imaged, it is essential to use an environmental chamber (we use one from LIS, Switzerland), controlling oxygen, humidity, CO2 and temperature. FRAP is performed using maximum laser intensity while imaging is done with the minimal laser power required (usually in the area of 10%, when fluorescence level is adequate). 
 Observe the cells with fluorescent light of the appropriate wavelength and select a cell expressing GFP using a 60X oil immersion lens. Ensure correct subcellular distribution. Occasionally, when expression levels are too high, the protein's localization may 'spill' to other compartments such as the nucleolus. Such cells should not be selected.</li>
 <li>Now set an imaging protocol: collect 3-5 frames before the photobleaching, then photobleach at euchromatin or heterochromatin (viewed as condensed GFP foci) and collect 90-120 frames after the photobleach, with 250-1000 ms intervals: H1e-GFP, 1000 ms, H1o and HP1-GFP, 250 ms (interval time changes according to the protein dynamics, where highly dynamic proteins require shorter interval time). We normally use 80-100% laser intensity for photobleaching with a laser pulse of 20-40 &mu;seconds (1-2 iterations), but these numbers can change according to the analyzed protein and the expression levels. When photobleaching is appropriate, you should observe a "black hole" in your GFP fluorescence. The black hole is gradually re-filled with fluorescence following recovery. Although the spinning disk can obtain up to around 60 images per second (with good fluorescence intensity and when zooming in on a single cell), we do not recommend using the system at such high speeds due to low image quality and potential increased phototoxicity.</li>
 <li>For a FLIP experiment, set up a different imaging protocol: collect 3-5 frames before bleaching, then start repeated bleaching on the same spot while collecting images. For H1e-GFP, bleach every 5 sec, for H1o-GFP, bleach every 2 sec, and for HP1-GFP bleach every 1 sec. Bleach and collect images repeatedly throughout the entire experiment.</li>
 <li>For either technique, repeat the process on 20-30 cells. For statistical purposes, repeat experiment 3 times or more, preferably on different days. In homogenous populations and correct settings, standard deviation is usually low (&lt; 5%). 
 For both FRAP and FLIP, the size and shape of the bleached region influences the recovery dynamics and must remain constant within an experiment. Also, when two cells are compared, identical protocols must be used and the cells must be analyzed sequentially on the same day as power laser and other conditions may fluctuate and may affect the outcome of the experiment.</li>
</ol>
4. FRAP and FLIP Data Analysis
<ol>
 <li>In all FRAP frames collected, measure the fluorescence intensity in the ROI (ROIb = bleached area), the background area (ROIbg), and the non-bleached nuclear area (ROInb) as a function of time before and after bleaching. When the bleached region is negligible the entire nucleus can be selected for normalization purposes.</li>
 <li>For every time point, normalize data according to the formula: (ROIb - ROIbg)/(ROInb - ROIbg) / (pbROIb - pbROIbg)/(pbROInb - pbROIbg), pb denotes pre-bleached. For pre-bleach images you should get a value of approximately 1. The first image after the bleach will indicate the Bleach Depth. Subtract the value from 1 for the actual Bleach Depth value. Repeat for every cell and average 20-30 cells from each experiment.</li>
 <li>In all FLIP frames collected, measure the fluorescence intensity in the non-bleached nuclear area, and the background area (ROInb = non-bleached area, 
 ROIbg = background). Calculation of FLIP data is similar to a FRAP curve, only the analyzed ROI (ROInb) should be different than the actual bleached region, which is not used for calculation: (ROInb - ROIbg)/(pbROInb - pbROIbg). It is also possible to use a neighboring cell (ROIn = neighbor cell) for normalization purposes: (ROInb - ROIbg)/(ROIn - ROIbg) / (pbROInb - pbROIbg)/(pbROIn - pbROIbg). 
 Following data collection, it is possible to fit the experimental data to computer simulation. This allows calculating, with a good proximity, the mobile fraction, the immobile fraction and the half-maximum. We will not discuss the mathematical and statistical aspects of FRAP analyses here and refer the reader to other excellent publications4-9. The bleach depth refers to the distance (on the y-axis) between the pre-bleach (100%) signal and the first image after bleach; the mobile fraction refers to the distance (on the y-axis) between the bleach depth and the recovered signal when the kinetics reaches a plateau, and the immobile fraction refers to the distance (on the y-axis) between the recovered signal and the pre-bleach (100%) signal (see Figures 1B and 2B). Beyond this analysis, there are good mathematical models to fit the data. For a single exponent, the equation  
	<img src="/files/ftp_upload/2696/eq0.jpg" alt="Equation" width="80" /> 
	where t is time, A is the mobile fraction, 1-A is the immobile fraction and koff is the dissociation constant, can be used to fit the data, and a direct estimate of the off rate of binding (koff) can be obtained, as well as for the parameter A, which can be used to calculate the association rate.</li>
</ol>
5. Representative Results: 
<img src="/files/ftp_upload/2696/2696fig1.jpg" alt="Figure 1" /> 
Figure 1. A and B show representative FRAP curves of HP1 (left), H1o (middle) and H1e (right) in R1 ES cells. For simplicity and clarity Figure 1A shows the raw data of a single cell before any normalization and calculation. The yellow curve corresponds to the bleached region, the purple curve corresponds to the non-bleached nuclear area (when the bleached region is negligible the entire nucleus can be selected for normalization purposes), and the red line corresponds to the background fluorescence, which is minimal in this case. Vertical arrow represents the bleach time. Normalized and averaged data is shown in Figure 1B. Note the slower recovery of H1 (blue) compared with HP1 (red). Also the H1e variant (dark blue) is slower than the H1o variant (light blue). Mobile and immobile fractions and Bleach Depth are indicated for HP1.
<img src="/files/ftp_upload/2696/2696fig2.jpg" alt="Figure 2" /> 
Figure 2. A and B show representative FRAP curves comparing euchromatin (left) with heterochromatin (right) of HP1 in R1 ES cells. Similarly to Figure 1, Figure 2A shows raw data of a single cell, yellow curve corresponds to the bleached region, purple curve corresponds to the non-bleached nuclear area, and the red line corresponds to the background fluorescence. Vertical arrow represents the bleach time. Normalized and averaged data is shown in Figure 2B. Note the slower recovery of heterochromatin (dark red) compared with euchromatin (light red). Mobile and immobile fractions and Bleach Depth are indicated for euchromatin.
<img src="/files/ftp_upload/2696/2696fig3.jpg" alt="Figure 3" /> 
Figure 3.. A typical FLIP experiment of H1o R1 ES cells is shown in Figure 3A (raw, un-normalized data) and B (normalized and averaged data). In this experiment the purple curve corresponds to the non-bleached nuclear area, the green line corresponds to a neighboring cell nucleus and the red line corresponds to the background fluorescence.

<ol>
	<li>Phair, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+nature+of+dynamic+protein-chromatin+interactions+in+vivo:+three-dimensional+genome+scanning+and+dynamic+interaction+networks+of+chromatin+proteins.">Global nature of dynamic protein-chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins.</a> Mol Cell Biol. 24, 6393-6402 (2004).</li><li>Meshorer, E., Girard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging chromatin in embyonic stem cells in StemBook. , (2008).</li><li>Meshorer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperdynamic+plasticity+of+chromatin+proteins+in+pluripotent+embryonic+stem+cells.">Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells.</a> Dev Cell. 10, 105-116 (2006).</li><li>Bancaud, A., Huet, S., Rabut, G., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence perturbation techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, photoactivation, photo conversion, and FLIP. , (2009).</li><li>Dundr, M., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+dynamics+of+nuclear+proteins+by+photobleaching.">Measuring dynamics of nuclear proteins by photobleaching.</a> Curr Protoc Cell Biol. Chapter 13, Unit 13-Unit 13 (2003).</li><li>Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+membrane+dynamics+and+reassembly+in+living+cells:+targeting+of+an+inner+nuclear+membrane+protein+in+interphase+and+mitosis.">Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis.</a> J Cell Biol. 138, 1193-1206 (1997).</li><li>Lenser, T., Weisshart, K., Ulbricht, T., Klement, K., Hemmerich, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+fluctuation+microscopy+to+reveal+3D+architecture+and+function+in+the+cell+nucleus.">Fluorescence fluctuation microscopy to reveal 3D architecture and function in the cell nucleus.</a> Methods Cell Biol. 98, 2-33 (2010).</li><li>Mueller, F., Mazza, D., Stasevich, T. J., McNally, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+and+kinetic+modeling+in+the+analysis+of+nuclear+protein+dynamics:+what+do+we+really+know.">FRAP and kinetic modeling in the analysis of nuclear protein dynamics: what do we really know.</a> Curr Opin Cell Biol. 22, 403-411 (2010).</li><li>Phair, R. D., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+modelling+approaches+to+in+vivo+imaging.">Kinetic modelling approaches to in vivo imaging.</a> Nat Rev Mol Cell Biol. 2, 898-907 (2001).</li><li>Poser, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+TransgeneOmics:+a+high-throughput+method+for+exploration+of+protein+function+in+mammals.">BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.</a> Nat Methods. 5, 409-415 (2008).</li><li>Sigal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+fluorescently+labeled+endogenous+protein+library+in+living+human+cells.">Generation of a fluorescently labeled endogenous protein library in living human cells.</a> Nat Protoc. 2, 1515-1527 (2007).</li><li>Cohen, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+proteomics+of+individual+cancer+cells+in+response+to+a+drug.">Dynamic proteomics of individual cancer cells in response to a drug.</a> Science. 322, 1511-1516 (2008).</li></ol>

No conflicts of interest declared.

Unlike most available techniques, which involve purified chromatin from cell populations or fixed cells, FRAP experiments follow changes in chromatin protein dynamics in living cells. We found chromatin protein dynamics to be a good indicator for chromatin plasticity. However, because it requires fusing the gene of interest with GFP, the addition of the fluorescent tag can interfere with the protein's function. Thus, prior to proceeding with FRAP, the fusion protein must be rigorously tested to ensure it has the same pro...

<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Sigma</td>
 <td>D5671</td>
 </tr>
 <tr>
 <td>Gelatin</td>
 <td>Merck</td>
 <td>1.04078</td>
 </tr>
 <tr>
 <td>Opti-MEM</td>
 <td>Gibco</td>
 <td>31985</td>
 </tr>
 <tr>
 <td>TransIT-LT1</td>
 <td>Mirus</td>
 <td>MIR2300</td>
 </tr>
 <tr>
 <td>8-well &mu;-Slides</td>
 <td>ibidi</td>
 <td>80826</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells at JoVE.com

Photobleaching Assays FRAP &amp; 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.

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 ...

photobleaching-assays-frap-flip-to-measure-chromatin-protein-dynamics

Research

JoVE Journal

Biology

27.1K Views.  The Hebrew University of Jerusalem. Мы опишем методы, включая фотообесцвечивания флуоресценции Восстановление после фотообесцвечивания (FRAP) и флуоресценции Потеря В фотообесцвечивания (FLIP) для контроля динамики хроматина белка в эмбриональных стволовых (ЭС) клеток. Хроматин динамики белков, к...

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

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

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 ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...