1. Telomere Length Protocol19
1. DNA isolation
Most DNA isolation methods may be used. Our Lab prefers Qiagen DNeasy Kit (#69506) for blood sources. Depending on the source of DNA, such as buccal or other sources, a different isolation method may be used.
2. Primers
All primers are diluted to a stock concentration of 100pmoles/&mu;l in PCR grade water and stored at -20 &deg;C until required. Working stocks of primers are made fresh and are stored at 20 &deg;C for a short period of time (A few months.) Standard primers were used for telomeres and &beta;-Globin, the SCG, as described in O'Callaghan et al. 20.
Primer sequences: 
Telo 1: 5'-CGGTTTGTTTGGGTTTGGGTTTGGGTT-TGGGTTTGGGTT-3'
Telo 2: 5'-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTT-ACCCT-3' 
SCG 1: 5'-GCTTCTGACACAACTGTGTTCACTAGC-3' 
SCG 2: 5'-CACCAACTTCATCCACGTTC-ACC-3'
Note: Primers are diluted to a working concentration of 10pmoles/&mu;l, prior to adding to reaction mix.
3. Serial Dilution of Genomic DNA
Serial dilutions of genomic DNA for Telo and Single Copy Gene (SCG) are created to generate a standard Ct value curve for the assay. The genomic DNA used was extracted from lymphocytes from blood samples. These dilutions are created by diluting DNA with PCR grade H20 to each concentration by a factor of 1.68 to yield the two sets of serial dilutions using the same genomic DNA. The starting concentrations have been optimized based on trial and error and may need to be adjusted when using different reagents, instruments, DNA from other species, cell types, and SCG used.
Note: Serial dilution using genomic DNA are made by mixing gently and waiting at least 15 min to one hour before taking DNA for the next dilution. Each dilution requires time for genomic DNA to dissociate. For long term storage, aliquot serial dilutions into PCR strips for storage at -80 &deg;C. Each PCR strip is thawed and used once to avoid freeze thaw cycles that may damage DNA.
4. Reaction setup for RT-PCR
Instrument used: Roche480 Light Cycler
Reagent: Roche Light cycler sybr-green
Dilute test DNA samples to a final concentration of 10 ng/&mu;l. We test concentration using Nanodrop or Qubit. We further dilute the DNA from 10ng/&mu;l to final 5ng/&mu;l. We do not test concentration again.
Reaction components:
<table border="1">
 <tbody>
 <tr>
 <td fo:width="1.5in">TELO</td>
 <td fo:width="1.5in">1x</td>
 <td fo:width=".25in" rowspan="6"> </td>
 <td fo:width="1.5in">SCG</td>
 <td fo:width="1.5in">1x</td>
 </tr>
 <tr>
 <td>H20</td>
 <td>6 &mu;l</td>
 <td>H20</td>
 <td>6 &mu;l</td>
 </tr>
 <tr>
 <td>Telo 1</td>
 <td>0.2 &mu;l</td>
 <td>SCG 1</td>
 <td>0.6 &mu;l</td>
 </tr>
 <tr>
 <td>Telo 2</td>
 <td>1.8 &mu;l</td>
 <td>SCG 2</td>
 <td>1.4 &mu;l</td>
 </tr>
 <tr>
 <td>Rxn mix</td>
 <td>10 &mu;l</td>
 <td>Rxn mix</td>
 <td>10 &mu;l</td>
 </tr>
 <tr>
 <td>DNA or serial dilution</td>
 <td>2 &mu;l</td>
 <td>DNA or serial dilution</td>
 <td>2 &mu;l</td>
 </tr>
 </tbody>
</table>
Note: Master Mix is made without DNA and with an excess of 5% to 10%.
Both Telo and SCG are run together on the same plate using the following program. To test for precision, at least three replicates of each sample and control should be run. Variations in the primers used may affect the results. Our protocol has been optimized to work as follows on the Roche480 Light Cycler and our primers.
Initial denature 
10 min denature 95 &deg;C 
Cycling 
95 &deg;C 10 sec hold 
60 &deg;C 5 sec hold 
72 &deg;C 11 sec hold and single acquisition 
Repeat 45 to 55 times as required
2. Quantitative Telomerase Detection (QTD) Protocol (Based on QTD Kit by Allied Biotech, Inc. Cat. No. MT3012)
Extract Preparation
<ol>
 <li>Pellet cells or tissue.</li>
 <li>Wash once with 1x PBS.</li>
 <li>Repellet and remove 1x PBS.*</li>
 <li>Resuspend the cell pellet in 200 &mu;l of 1 x Lysis Buffer for every 105-106 cells or 40-100 mg of tissue.</li>
 <li>Incubate on ice for 30 min.</li>
 <li>Spin down the sample at 12,000 x g for 30 min at 4 &deg;C.</li>
 <li>Transfer 160 &mu;l of the supernatant into a fresh tube and determine protein concentration.</li>
 <li>Aliquot and quick-freeze the remaining extract on dry ice/ethanol, store at -80 &deg;C.</li>
</ol>
*In this condition, telomerase in frozen cells or tissues is stable for at least a year. When thawed for use, resuspend the cells immediately in 1 x Lysis Buffer.
2.1. Assay Controls
Heat inactivation control: For each sample, heat treat an additional control extract by incubating at 85 &deg;C for 10 min prior to telomerase activity assay.
Standard curve for TSR control template: Perform Quantitative Real-Time PCR using dilutions of TSR, an oligonucleotide with a similar sequence to telomere primers included with the kit to generate a standard curve.
<ol>
 <li>Prepare 1:5 serial dilutions of the stock TSR concentration (0.5 amoles/&mu;l) with Lysis Buffer</li>
 <li>Perform the telomerase detection standard assay using 1 &mu;l of each TSR dilution, including the stock concentration. These dilutions can be stored at 4 &deg;C for at least 2 weeks.</li>
</ol>
2.2. Telomerase Activity Assay using QTD Real-Time PCR
<ol>
 <li>For each sample, add the following to a PCR tube or thin-wall PCR plate (total volume 25.0 &mu;l):
 <ul>
 <li>12.5 &mu;l of 2 x QTD Premix</li>
 <li>1.0 &mu;l of Cell or Tissue Extract</li>
 <li>11.5 &mu;l of PCR Qualified Water</li>
 </ul>
 </li>
 <li>Mix thoroughly</li>
 <li>Program Real-Time PCR Detection Systems according to the below program: 
 - 25 &deg;C for 20 min (telomerase reaction) 
 - 95 &deg;C for 10 min (PCR initial activation step) 
 35-40 cycles of: 
 - 95 &deg;C for 30 sec (denaturation) 
 - 60 &deg;C for 30 sec (annealing) 
 - 72 &deg;C for 30 sec (extension)</li>
 <li>Place PCR tubes in the thermal cycler and start the cycling program.</li>
 <li>Collect the threshold cycle or CT value after cycles finish. The threshold cycle is the cycle at which a statistically significant increase of &Delta;Rn is first detected.</li>
</ol>
2.3. Data Analysis
<ol>
 <li>Generate a standard curve using the CT readings and compare telomerase activity.</li>
</ol>

No conflicts of interest declared.

Telomere length provides a unique cellular marker to study the stressed and aging cell and offers insights into the mechanisms of aging. Since its role in aging had first been suggested, a multitude of studies have been done relating telomere length to age, longevity, age-related disease, cancers, and stress. For decades, the gold standard of telomere length measurements was terminal restriction fragment analysis using Southern hybridization. However, recently an affordable, quick, and easy to perform method by Ca...

Telomeres are repeating DNA hexamer (TTAGGG) sequences found at the ends of chromosomes. In each cell replication, these chromosome ends are shortened. If they become too short, chromosomes can undergo telomere end fusions, aberrant recombination, and degradation. Thus sufficient telomere length maintenance plays a major role in chromosome stability and cell protection 38. Telomere length maintenance is also crucial for genes found near the ends of chromosomes, since DNA replication cannot continue to the very end of chromosomes 1-2. Consequently, prevention of telomere shortening may improve cell stability.
Numerous studies have reported that telomere length is correlated with an organism's longevity and diseased state, such as in cancer 3-4, diabetes 5, and cardiovascular disease 6,7. Additionally, shortened telomeres have been associated with excessive stress or an unhealthy lifestyle 8, perhaps by promoting premature cell aging and death 9. On the other hand, some studies have found that there is no significant difference between telomere length and longevity and age related disease 39, 40, 41.
One of the cell's innate mechanisms of protection from telomere shortening is by activating its own enzyme telomerase reverse transcriptase (TERT). This enzyme and its subunit, telomerase RNA (TERC), a non-coding RNA, use the telomere as a template to add telomere repeats to chromosome ends 10. Although telomerase activity is absent from several types of cells and other mechanisms are involved in telomere length maintenance, increased telomerase activity is correlated with increased telomere length. Telomerase activation has been established as one of the mechanisms by which cells respond to damage and stress and avoid premature senescence and death. For example, longer telomeres were demonstrated in a population of Ashkenazi Jews with exceptional lifespan 11, mutations in TERC or TERT have been shown to contribute to fatal disease 12, and epigenetic regulation of telomerase has been shown to have an effect on age-related disease 13. Since its discovery, telomere length has been proposed as a biomarker for health status in various animal models as well as in humans, but these early studies were difficult to widely replicate because the method used was tedious, long and expensive. In 2002 and further tuned in 2009, Cawthon proposed and demonstrated a new, accurate, fast and simple PCR based protocol to assess telomere length and further investigate its role in various aspects of cell biology, aging and disease 19.
Choosing the correct telomere measuring method for a study is essential. Currently, there are various methods used to measure telomere length, each with its own advantages and disadvantages. Traditionally, telomere length is measured using Southern Blot analysis of terminal restriction fragments (TRFs), which involves: a. digesting the DNA with restriction enzymes that do not cut in telomere repeats in order to obtain TRFs, b. Southern Blot of these TRFs is done by determining the mean TRF length using a telomeric probe 14, 37. Although this method is highly accurate with a small coefficient of variation, is a direct measure, and can be advantageous for measuring length distribution, TRF analysis is costly, labor intensive and requires at least 3 &mu;g of DNA. This method is also insensitive to short telomeres and length determination can be confounded by subtelomeric DNA, which can be detected by the probe due the (TTAGGG)n like sequences they contain 15, 42.
Another highly accurate method in measuring telomere length is Single Telomere Elongation Length Analysis (STELA), a single molecule PCR based method that only requires a small amount of DNA. In this method, primers are made to recognize the G-rich overhang at the end of chromosomes and to bind to a unique subtelomeric sequence on one chromosome, which amplifies the telomere of a specific chromosome. The resulting amplification is then visualized by Southern Blot. This method has the advantage of being highly accurate and is able to detect short outlier telomeres 16. Unfortunately, since not all chromosomes have G-rich ends and a usable subtelomeric sequence, telomere length can only be measured on specific chromosomes and these measurements may not represent the length of all the telomeres in the cell 15.
Another method that has been practiced is the use of Peptide Nucleic Acid (PNA) probes to detect telomere length. Quantitative Florescence in situ Hybridization (Q-FISH) allows us to visualize telomeres during metaphase, where the staining of telomeres with a PNA probe in proportion to their size permits the comparison of telomeres between specific chromosomes. Although this method can also be used for cells in interphase, here the telomere length for distinct chromosomes cannot be detected, which introduces a limitation when measuring telomere length in senescent or infrequently dividing cells 17. Flow FISH instead uses flow cytometry and is currently the most sensitive method for measuring telomere length of blood cells in the clinical setting, but requires highly skilled technicians 18.
Currently, the standard method for measuring average telomere length in our lab takes advantage of the precision, sensitivity, and ease of quantitative real-time PCR. This method was made possible for measuring telomere length by the development of novel primers that avoid the synthesis of primer dimer-derived products, which would have otherwise been produced in a standard assay due to the repeating nature of telomeres. For this assay, the measurement of telomere length is represented by the T/S ratio, the telomere repeat copy number to single-copy gene number. Since there is a direct proportional relationship between the telomere length and the number of labeled telomere primers binding to the DNA during the beginning stages of PCR, the T/S ratio is directly proportional to telomere length. The T/S ratio is measured by comparing the difference in Ct, the fractional cycle number at which the sample's accumulated florescence crosses a threshold that is several standard deviations above the baseline florescence, between samples with telomere primers and SCG primers 19. This method has been criticized for its indirect measurement of telomere length, which can lead to inaccurate measurements, for example, in the case of chromosome duplications or copy number variations 15. Also, comparison between studies is often difficult, but standard oligomers have been developed to measure absolute telomere length 20. This method was furthered improved upon by Cawthon, using a monochrome multiplex qPCR assay. In this assay, the PCR was run at lower temperatures for the first few cycles to avoid primer-dimer binding and the telomere and control gene were analyzed in the same PCR tube to further avoid error. The resulting telomere length measurements strongly correlated with telomere length measured by Southern blot analysis of TRFs and had higher accuracy 15, 19, 36. At the moment, qRT-PCR is the only convenient method available for testing large sample sizes and only requires a small amount of DNA to carry out. A crucial part of this method is comprehensive quality control measures, so that when it's done properly, this method can provide valuable comparative information about telomere length. Additionally, this method had been adapted for use beyond leukocytes for measuring telomere length in a variety of different tissues 42.
Additionally, in order to further understand the biology behind telomere length maintenance and interactions between the two opposing effects (i.e. telomere shortening during replication and elongation by the telomerase enzyme), we accompanied the telomere length method with an additional assay that measures telomerase activity.
For this purpose, we used a Telomeric Repeat Amplification Protocol, an in vitro assay. Briefly, lysed, preserved, enzymatically active cells synthesized telomeric repeats onto an oligonucleotide substrate using telomerase, and the products were amplified using PCR in the presence of SYBR Green. The results were then analyzed by comparing sample and control Ct threshold values. Since telomerase is a heat-sensitive enzyme, an additional heat treated control is run alongside each sample 21.
While measuring telomere length can provide valuable insight into the possible role of telomere length in the pathophysiology of aging and disease, telomere length is not a static property and more information is needed to understand telomere function. Telomerase activity studies can add information about telomere length regulation mechanisms. For example, the controlled activation of telomerase can maintain telomere length and cell proliferation, but the uncontrolled activation of telomerase can result in cancer. These two inexpensive and straight forward methods combined provided us not only with stronger evidence towards a relationship between telomere length and cell stability, but also further insight into the possible mechanisms of telomere shortening and recovery. With these methods we hope to further elucidate the cell's response to aging and various states of cell proliferation with the ultimate goal of a better understanding of the central mechanisms of cell biology.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Quantitative Telomerase Detection </td>
 <td>Allied Biotech </td>
 <td>MT3012 </td>
 </tr>
 <tr>
 <td>Qiagen DNeasy Kit</td>
 <td>Qiagen</td>
 <td>69506</td>
 </tr>
 <tr>
 <td>LightCycler 480 SYBR Green I Master, Ready-to-use hot start reaction mix for SYBR Green I-based real-time PCR using the LightCycler 480 Instrument</td>
 <td>Roche Diagnostics</td>
 <td>4707516</td>
 </tr>
 </tbody>
</table>

telomere length and telomerase activity; a yin and yang of cell senescence

Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

An example of a telomere length qRT-PCR assay is shown in Figure 1. On the top left panel, samples labeled in red and green represent the location of the tested subjects on the 96-well plate. On the top right panel, the amplification curve is demonstrated. Each subject is tested by two assays (Telomere and Single Copy Gene), which is done in triplicates. Due to different DNA copy numbers produced in the two assays of selected individuals, the number of cycles until the florescence detection reache...

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Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

telomere-length-telomerase-activity-yin-yang-cell

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46.4K ビュー.  Albert Einstein College of Medicine. 、正確な短い、洗練された安価な方法は、定量RT-PCRを使用して、複数の組織や種のテロメアの長さを評価することが記載されている。また、テロメアの長さのために相補的なバックボーン試験としてテロメラーゼ活性を評価するための簡単​​なアッセイについて説明する。

ビデオ: Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven. In many cases membrane-spanning proteins have evolved highly sophisticated mechanisms of action.1-3 One way in which membrane proteins can modulate their structures and functions is by direct and specific contact of hydrophobic helices, forming structured transmembrane oligomers.4,5 Much recent work has focused on the distribution of amino acids preferentially found in the membrane environment in comparison to aqueous solution and the different intermolecular forces that drive protein association.6,7 Nevertheless, studies of molecular recognition at the transmembrane domain of proteins still lags behind those of water-soluble regions. A major hurdle remains: despite the remarkable specificity and affinity that transmembrane oligomerization can achieve,8 direct measurement of their association is challenging. Traditional methodologies applied to the study of integral membrane protein function can be hampered by the inherent insolubility of the sequences under examination. Biophysical insights gained from studying synthetic peptides representing transmembrane domains can provide useful structural insight. However, the biological relevance of the detergent micellar or liposome systems used in these studies to mimic cellular membranes is often questioned; do peptides adopt a native-like structure under these conditions and does their functional behaviour truly reflect the mode of action within a native membrane? In order to study the interactions of transmembrane sequences in natural phospholipid bilayers, the Langosch lab developed ToxR transcriptional reporter assays.9 The transmembrane domain of interest is expressed as a chimeric protein with maltose binding protein for location to the periplasm and ToxR to provide a report of the level of oligomerization (Figure 1).
In the last decade, several other groups (e.g. Engelman, DeGrado, Shai) further optimized and applied this ToxR reporter assay.10-13 The various ToxR assays have become a gold standard to test protein-protein interactions in cell membranes. We herein demonstrate a typical experimental operation conducted in our laboratory that primarily follows protocols developed by Langosch. This generally applicable method is useful for the analysis of transmembrane domain self-association in E. coli, where &#946;-galactosidase production is used to assess the TMD oligomerization propensity. Upon TMD-induced dimerization, ToxR binds to the ctx promoter causing up-regulation of the LacZ gene for &#946;-galactosidase. A colorimetric readout is obtained by addition of ONPG to lyzed cells. Hydrolytic cleavage of ONPG by &#946;-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP) (Figure 2).

An efficient procedure to assess the oligomerization propensity of single-pass transmembrane domains (TMDs) is described. Chimeric proteins consisting of the TMD fused to ToxR are expressed in an E. coli reporter strain. TMD-induced oligomerization causes dimerization of ToxR, activation of transcription and production of the reporter protein, -galactosidase.

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven.  In many cases ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...