eoe sub articles

EoE Sub Articles

1. Preparation of Media and Reagents
<ol>
	<li>Thaw the basement membrane matrix coating for culture plates at 4 &#176;C (do not allow the basement membrane matrix to warm up, or it will solidify). Make 1 mL aliquots and store them at -20 &#176;C or -70 &#176;C.</li>
	<li>Reconstitute basic fibroblast growth factor (bFGF) in sterile phosphate-buffered saline (PBS) at 10 &#956;g/mL and make 10 &#956;L aliquots. Store them at 4 &#176;C.</li>
	<li>To a new, unopened, 500 mL bottle of DMEM/F12 with glutamine, add B27 (10 mL), N2 (5 mL), and penicillin-streptomycin (5 mL). Place 50 mL of this neural stem cell (NSC) media in a conical tube and add 10 &#956;L of 10 &#956;g/&#956;L (2 &#956;g/mL final) bFGF. Store the NSC (+) bFGF media at 4 &#176;C.</li>
	<li>To make (-) bFGF media, use the same recipe as described in step 1.3 but do not add bFGF. This media will be used to differentiate NSCs to neurons and to maintain neuronal cultures after differentiation.</li>
</ol>
2. Lentiviral Constructs
NOTE: Before beginning work with lentiviral constructs, ensure that the lab has been approved to use biosafety level 2 (BSL-2) agents. Furthermore, BSL-2 culture hoods, personal protective equipment (PPE), and disposal methods must be used when working with lentiviral vectors.
<ol>
	<li>Obtain tau constructs packaged into lentivirus from a preferred source.</li>
</ol>
3. Culturing Human Neural Stem Cells
NOTE: NSCs are typically seeded at 100,000&#8211;150,000 cells/cm2 and most commercially available NSCs are sold as 1 x 106 cells/vial. This protocol has been optimized for 10 cm cell culture dishes (although other sizes of dishes may be used); therefore, if commercially available NSCs are being used, the NSCs may need to be expanded by first being cultured in six-well dishes in order to result in enough cells to seed 10 cm dishes. This protocol can alternatively be adapted for a variety of cell culture dish sizes.
<ol>
	<li>For the preparation of cell culture plates, remove one aliquot of frozen basement membrane matrix coating for cell culture plates and allow it to thaw at 4 &#176;C (aliquots can be placed at 4 &#176;C overnight the day before the cells are to be seeded). Add 385 &#956;L of basement membrane matrix coating to 5 mL of DMEM/F12 media + penicillin-streptomycin. 
	NOTE: 5 mL of DMEM/F12 media + penicillin-streptomycin is enough to coat a single 10 cm dish (1 mL of this basement membrane matrix coating solution is sufficient to coat one well of a six-well dish). Alternatively, if the cells are to be fixed and immunostained, or stained for thioflavin, culture cells on glass coverslips in 24-well culture dishes.
	<ol>
		<li>Keep the media and the matrix coating cold before and while adding them to culture dishes. Add the basement membrane matrix coating to culture dishes and place the dishes in an incubator (at 37 &#176;C) for 1 h. For optimal coating, do not incubate the basement membrane matrix for more than or less than 1 h. After 1 h, aspirate the basement membrane matrix coating from the cell cultures dishes. Make sure this coincides with the NSCs being ready to be plated. 
		NOTE: If cells are not being thawed from frozen stocks, skip step 3.2 and continue with step 3.3.</li>
	</ol>
	</li>
	<li>If cells are being thawed from frozen NSC stocks, take a vial of frozen cells and warm it in a water bath heated to 37 &#176;C by moving the vial back and forth in the water. Once thawed, spray the vial with 70% ethanol and place it into a cell culture hood. Transfer the cells to ~10 mL of DMEM/F12 + penicillin-streptomycin media and centrifuge the tube at room temperature at 1,000 x g for 5 min. Aspirate the media and resuspend the cells in NSC media.</li>
	<li>Dilute the cells in NSC media to obtain the appropriate seeding density for the cell culture vessel that is being used. 
	NOTE: For example, if NSCs are being plated onto a six-well plate&#8212;one well on a six-well culture dish has a surface area of 9 cm2&#8212;900,000 to 1,350,000 cells are required to seed. So, one vial containing 1,000,000 cells can be resuspended in 2 mL of media and added to a single well of a six-well dish.</li>
	<li>Add enough cells suspended in NSC media to seed the basement membrane matrix-coated culture dishes (100,000 cells/cm2) and change the media every other day (if using a frozen stock, change the media the day after plating and every other day thereafter). Grow the cells until they are 75%&#8211;80% confluent. 
	NOTE: The cells will likely reach 75%&#8211;80% confluence shortly after plating, but this may take longer if frozen stocks are used.</li>
	<li>Once the NSCs are 75%&#8211;80% confluent, begin neuronal differentiation by removing the NSC (+) bFGF media and replacing it with NSC (-) bFGF media. Culture the cells in this media (replacing the media every other day) for at least 4 weeks. 
	NOTE: The cells will continue to divide for a few days following the withdrawal of bFGF; therefore, cells may reach 90&#8211;100% confluence.</li>
	<li>After culturing for 4 weeks, the cells will have achieved a neuronal fate and will be ready for the treatment of lentivirus.</li>
</ol>
4. Transduction and Maintenance of Neuronal Cultures
<ol>
	<li>To transduce neurons with lentivirus, use a titer count of 3.4 x 105 transducible units/cell (a 100% confluent 10 cm dish will contain ~10 million neurons). Dilute the transducible units to the necessary concentration in cell culture media and add them to the cells (use the normal volume of media for the cell culture dish). Two days after adding the lentivirus, wash the cells 1x with fresh (-) bFGF media (without lentivirus) and continue culturing in (-) bFGF media as usual.</li>
	<li>For the post-lentiviral treatment, feed the transduced neurons by changing the cell culture media ([-] bFGF media) every other day. Maintain the cells for ~8 weeks after transduction. Routinely visualize the cells under a light microscope to ensure viability. 
	NOTE: Sparseness or dendritic breakages are signs that the cells are no longer viable.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm culture dishes</td>
			<td>Thermofisher</td>
			<td>12556002</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Biopioneer</td>
			<td>CNT-50</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol in spray bottle</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermofisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Basement membrane matrix (Matrigel)</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic FGF</td>
			<td>Biopioneer</td>
			<td>HRP-0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12 culture media with glutamine</td>
			<td>Thermofisher</td>
			<td>10565042</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (50% concentration or higher)</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Human neural stem cells</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentiviral vectors</td>
			<td>Various sources</td>
			<td>custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermofisher</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermofisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Thermofisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell culture hood</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

lentiviral-mediated transduction of human neurons for tau protein aggregation analysis

Source: Aulston, B., et al. <a href="https://app.jove.com/v/59433">An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons.</a>&#160;
This video demonstrates the process of transducing human neuronal cells with a lentivirus encoding a mutant human tau protein tagged with yellow fluorescent protein (YFP) to study protein aggregation. It outlines the steps from viral entry to mutant tau expression, culminating in the formation of visible cytoplasmic tau aggregates in the neurons.

Lentiviral-Mediated Transduction of Human Neurons for Tau Protein Aggregation Analysis

Source: Aulston, B., et al. An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons.&#160;
This video ...

Begin with an adherent culture of mature human neurons in a matrix-coated plate.
Add lentiviral vectors encoding a mutant human tau protein fused to a yellow fluorescent protein (YFP) reporter.
The lentivirus attaches to specific neuronal cell surface receptors, enabling viral-host membrane fusion and the release of viral RNA and enzymes.
The viral RNA is reverse-transcribed into DNA.
Viral integrase transports the DNA into the nucleus, facilitating integration into the host genome, and leading to the production of YFP-tagged mutant tau protein.
Over time, the mutant tau forms cytoplasmic aggregates in the transduced cells.
Wash the cells with fresh media to remove non-internalized viral particles.
Continue incubation with regular media changes to maintain neuronal viability and promote protein aggregation.
Visualize the neurons under a microscope.
Successfully transduced neurons exhibit cytoplasmic aggregates of fluorescent YFP-tagged mutant tau protein.

lentiviral-mediated-transduction-human-neurons-for-tau-protein

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

32 次观看. Source: Aulston, B., et al. An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons.  This video demonstrates the process of transducing human neuronal cells with a lentivirus encoding a mutant human tau protein tagged with yellow fluorescent protein (YFP) to study protein aggregation. It outlines the steps from viral entry to mutant tau expression, culminating in the formation of visible cytoplasmic tau aggregates in the neurons. 

视频: Lentiviral-Mediated Transduction of Human Neurons for Tau Protein Aggregation Analysis - 概念

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, the data obtained from rodent islets are often not fully reproduced in or applicable to human islets due to well-known differences in islet structure and function between the species. Currently, techniques that are available to manipulate gene expression of human islets are very limited. Introduction of transgene into intact islets by adenovirus, plasmid, and oligonucleotides often suffers from low efficiency and high toxicity. Low efficiency is especially problematic in gene downregulation studies in intact islets, which require high efficiency. It has been known that enzymatically-dispersed islet cells reaggregate in culture forming spheroids termed pseudoislets. Size-controlled reaggregation of human islet cells creates pseudoislets that maintain dynamic first phase insulin secretion after prolonged culture and provide a window to efficiently introduce lentiviral short hairpin RNA (shRNA) with low toxicity. Here, a detailed protocol for the creation of human pseudoislets after lentiviral transduction using two commercially available multiwell plates is described. The protocol can be easily performed and allows for efficient downregulation of genes and assessment of dynamism of insulin secretion using human islet cells. Thus, human pseudoislets with lentiviral mediated gene modulation provide a powerful and versatile model to assess gene function within human islet cells.

A protocol to create gene modified human pseudoislets from dispersed human islet cells that are transduced by lentivirus carrying short hairpin RNA (shRNA) is presented. This protocol utilizes readily available enzyme and culture vessels, can be performed easily, and produces genetically modified human pseudoislets suitable for functional and morphological studies.

伦蒂病毒介导基因在低附着板中制备的人类伪性沉默

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, ...

科研

JoVE Journal

遗传学

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

The tau aggregation assay described in this manuscript mimics the anticipated features of in vivo tau misfolding and aggregation.

在体外陶氏蛋白聚集与药物筛选研究方法

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...

神经科学

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

Lentiviral-Mediated Transduction of Human Neurons for Tau Protein Aggregation Analysis

成績單

Lentiviral-Mediated Transduction of Human Neurons for Tau Protein Aggregation Analysis

伦蒂病毒介导基因在低附着板中制备的人类伪性沉默

在体外陶氏蛋白聚集与药物筛选研究方法

体外聚集测定使用tau蛋白的蛋白质