Name: production and use of lentivirus to selectively transduce primary oligodendrocyte precursor cells for in vitro myelination assays
Uploaded: 2015-01-12T14:00:00
Description: Watch this Scientific Journal Video about Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays at JoVE.com

This work was supported by the Australian National Health and Medical Research Council (NHMRC fellowship #454330 to JX, project grant #628761 to SM and APP1058647 to JX), Multiple Sclerosis Research Australia (MSRA #12070 to JX), the University of Melbourne Research Grant Support Scheme and Melbourne Research CI Fellowship to JX as well as Australia Postgraduate Scholarships to HP and AF.&#160;We would like to acknowledge the Operational Infrastructure Scheme of the Department of Innovation, Industry and Regional Development, Victoria Australia.

NOTE: All animals used for this study were of mixed sex and bred at the Animal Facilities of the Department of Anatomy &#38; Pathology and The Florey Institute of Neuroscience and Mental Health Research at the University of Melbourne. All animal procedures were approved by Animal Experimentation Ethics Committees at the University of Melbourne.
1. Cloning of 2K7 Lentivector
<ol>
	<li>Before cloning the gene of interest into the 2K7 lentiviral vector, subclone the gene into the pENTR vector (...

<ol>
	<li>Baumann, N., Pham-Dinh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+oligodendrocyte+and+myelin+in+the+mammalian+central+nervous+system.">Biology of oligodendrocyte and myelin in the mammalian central nervous system.</a> Physiol Rev. 81 (2), 871-927 (2001).</li><li>Watkins, T. A., Emery, B., Mulinyawe, S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+stages+of+myelination+regulated+by+gamma-secretase+and+astrocytes+in+a+rapidly+myelinating+CNS+coculture+system.">Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system.</a> Neuron. 60 (4), 555-569 (2008).</li><li>Lee, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDGAs+interact+selectively+with+neuroligin-2+but+not+other+neuroligins+to+regulate+inhibitory+synapse+development.">MDGAs interact selectively with neuroligin-2 but not other neuroligins to regulate inhibitory synapse development.</a> Proc Natl Acad Sci U S A. 110 (1), 336-341 (2013).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF+exerts+contrasting+effects+on+peripheral+myelination+of+NGF-dependent+and+BDNF-dependent+DRG+neurons.">BDNF exerts contrasting effects on peripheral myelination of NGF-dependent and BDNF-dependent DRG neurons.</a> J Neurosci. 29 (13), 4016-4022 (2009).</li><li>Chan, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF+controls+axonal+receptivity+to+myelination+by+Schwann+cells+or+oligodendrocytes.">NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.</a> Neuron. 43 (2), 183-191 (2004).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-Derived+Neurotrophic+Factor+Promotes+Central+Nervous+System+Myelination+via+a+Direct+Effect+upon+Oligodendrocytes.">Brain-Derived Neurotrophic Factor Promotes Central Nervous System Myelination via a Direct Effect upon Oligodendrocytes.</a> Neurosignals. 18 (3), 186-202 (2010).</li><li>Lundgaard, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuregulin+and+BDNF+induce+a+switch+to+NMDA+receptor-dependent+myelination+by+oligodendrocytes.">Neuregulin and BDNF induce a switch to NMDA receptor-dependent myelination by oligodendrocytes.</a> PLoS Biology. 11 (12), e1001743 (2013).</li><li>Kleitman, N., W, P. M., Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tissue culture methodes for the study of myelination. , (1991).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+signal-regulated+kinase+1/2+signaling+promotes+oligodendrocyte+myelination+in+vitro.">Extracellular signal-regulated kinase 1/2 signaling promotes oligodendrocyte myelination in vitro.</a> J Neurochem. 122 (6), 1167-1180 (2012).</li><li>Wong, A. W., Xiao, J., Kemper, D., Kilpatrick, T. J., Murray, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendroglial+expression+of+TrkB+independently+regulates+myelination+and+progenitor+cell+proliferation.">Oligodendroglial expression of TrkB independently regulates myelination and progenitor cell proliferation.</a> The Journal of Neuroscience. 33 (11), 4947-4957 (2013).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning,+Characterization+and+Expression+of+miR-15a-3p+and+miR-15b-3p+in+Dairy+Cattle.">Molecular cloning, Characterization and Expression of miR-15a-3p and miR-15b-3p in Dairy Cattle.</a> Molecular and Cellular Probes. , (2014).</li><li>Emery, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+gene+regulatory+factor+is+a+critical+transcriptional+regulator+required+for+CNS+myelination.">Myelin gene regulatory factor is a critical transcriptional regulator required for CNS myelination.</a> Cell. 138 (1), 172-185 (2009).</li><li>Murai, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+receptor+TLX+stimulates+hippocampal+neurogenesis+and+enhances+learning+and+memory+in+a+transgenic+mouse+model.">Nuclear receptor TLX stimulates hippocampal neurogenesis and enhances learning and memory in a transgenic mouse model.</a> Proc Natl Acad Sci U S A. 111 (25), 9115-9120 (2014).</li></ol>

Myelination of axons is a crucial process for the optimal function of both the central and peripheral nervous systems of vertebrates. The generation and maintenance of myelinated axons is a complex and coordinated process involving molecular interactions between neuronal, glial (from Schwann cells or oligodendrocytes) and extra cellular matrix proteins. The significance and applicability of this protocol is that it allows manipulation of proteins in one specific cell type within the mixed co-culture settings. As multiple...

Myelination of axons is crucial for the fast and efficient transmission of action potentials in both the central and peripheral nervous systems. Specialized cells, Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system, wrap around and ensheathe axons in myelin, effectively insulating the nerve and facilitating saltatory conduction1. The process of myelination can be studied in vitro using retinal ganglion neurons2, engineered nanofibers3, or dorsal root ganglion neurons co-cultured with either Schwann cells4 or oligodendrocytes5-7. The in vitro myelination assay is an established model for studying nervous system myelination and it replicates many of the fundamental processes that occur during myelination in vivo5-8. The assay involves the coculture of purified populations of Dorsal Root Ganglion (DRG) neurons, with OPCs (for CNS myelination) or Schwann cells (for PNS myelination). Under specific conditions these myelinating cells ensheathe DRG axons in the ordered, ultra structurally verified, multi-lamellar sheet of insulating plasma membrane that express the same complement of myelin specific proteins present in vivo.
The most commonly used cell model of studying CNS myelination in vitro is the co-cultures of DRG neurons and OPCs, which have been successfully used to study the effect that exogenous factors such as the neurotrophins exert on CNS myelination in vitro5,6. Exogenous factors such as growth factors or small molecule pharmacological inhibitors have been widely used to study the role of signaling pathways in myelination using the DRG-OPC coculture model7,9. However, in the mixed co-culture settings that contain both the neurons and oligodendrocytes, it remained formally possible that either the growth factors or the pharmacological inhibitors could have exerted effects upon both the DRG neurons and oligodendrocytes (OL). This does offer the ability to specifically dissect the roles that the proteins expressed only by DRGs or oligodendroglia exerts upon myelination using this dual cell system. To unequivocally confirm that the signaling pathway in oligodendroglial directly regulates myelination, lentiviral transduction of OPCs, prior to seeding onto DRG neurons for the in vitro myelination assay, has proven to be an elegant way to overexpress both wild-type and mutant proteins, as well as knockdown expression of constitutively expressed proteins by oligodendrocytes. Thus this approach offers an avenue to specifically interrogate and manipulate signaling pathways within oligodendrocytes for studying myelination9,10.
In this paper, we report methods that we have developed to overexpress a protein of interest selectively in oligodendrocytes via a lentiviral approach for studying myelination in vitro. The technique begins with the generation of expression vectors containing the gene of interest, be it in a wild type, constitutively active or dominant negative form which are then subsequently cloned into the pENTR vector (pENTR L1-L2 pENTR4IRES2GFP). This vector (containing the gene of interest), the CMV promotor donor (pENTR L4-R1 pENTR-pDNOR-CMV) and the 2K7 lentivector are combined in an enzyme reaction to produce a 2K7 vector containing CMV promoter, the gene of interest, an internal ribosomal entry site and GFP (Figure 1). This Gateway cloned 2K7 construct combined with the PMD2.G virus envelope and the pBR8.91 virus package can be co-transfected into HEK293T cells to generate lentivirus that can subsequently be used to transduce OPCs. Once infected with the lentivirus the OPCs express a high level of the protein of interest. These OPCs can then be seeded onto DRG neuron cultures and the effect that expression of high levels of the desired protein exerts on myelination can be interrogated. The co-cultures are assessed for myelin protein expression by western blot analysis and visualized for the formation of myelinated axonal segments by immunocytochemistry.

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			<td height="21" style="height:21px;width:413px;">Item</td>
			<td style="width:148px;">Manufacturer</td>
			<td style="width:99px;">Catalog #</td>
			<td style="width:183px;">Notes</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">2K7 lentivector&#160;</td>
			<td></td>
			<td></td>
			<td>Kind gift from Dr Suter9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Fluoro-2&#8242;-deoxyuridine</td>
			<td>Sigma-Aldrich</td>
			<td>F0503-100mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat anti-mouse IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>115545205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 goat anti-rabbit IgG (H+L)</td>
			<td>Life Technologies</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 goat anti-mouse IgG (H+L)</td>
			<td>Life Technologies</td>
			<td>A11005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 goat anti-rabbit IgG (H+L)</td>
			<td>Life Technologies</td>
			<td>A11012</td>
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			<td>A9518-5G</td>
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			<td height="21" style="height:21px;">B27 - NeuroCul SM1 Neuronal Supplement</td>
			<td>Stem Cell Technologies&#160;</td>
			<td>5711</td>
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		<tr height="21">
			<td height="21" style="height:21px;">BDNF (Human)</td>
			<td>Peprotech</td>
			<td>PT450021000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Biotin (d-Biotin)</td>
			<td>Sigma Aldrich</td>
			<td>B4639</td>
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			<td height="21" style="height:21px;">Bradford Reagent</td>
			<td>Sigma Aldrich</td>
			<td>B6916-500ML&#160;&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma Aldrich</td>
			<td>A4161</td>
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			<td height="21" style="height:21px;">Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378-100G</td>
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			<td height="21" style="height:21px;">CNTF</td>
			<td>Peprotech</td>
			<td>450-13020</td>
			<td></td>
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			<td height="21" style="height:21px;">DAKO fluoresence mounting media</td>
			<td>DAKO</td>
			<td>S302380-2</td>
			<td></td>
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			<td height="21" style="height:21px;">DMEM, High Glucose, Pyruvate, no Glutamine</td>
			<td>Life Technologies</td>
			<td>10313039</td>
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			<td height="21" style="height:21px;">DNase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025-375KU</td>
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			<td height="21" style="height:21px;">DPBS</td>
			<td>Life Technologies</td>
			<td>14190250</td>
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			<td height="21" style="height:21px;">DPBS, calcium, magnesium</td>
			<td>Life Technologies</td>
			<td>14040182</td>
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			<td height="21" style="height:21px;">EBSS</td>
			<td>Life Technologies</td>
			<td>14155063</td>
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			<td height="21" style="height:21px;">EcoRI-HF</td>
			<td>NEB</td>
			<td>R3101</td>
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			<td height="21" style="height:21px;">Entry vectors for promoter and gene of interest</td>
			<td></td>
			<td></td>
			<td>Generate as per protocols 1-2</td>
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			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>12003C</td>
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			<td>F6886-50MG</td>
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			<td>Sigma-Aldrich</td>
			<td>G7528</td>
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			<td>Chem Supply</td>
			<td>GL010-500M</td>
			<td>See stock solutions</td>
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			<td height="21" style="height:21px;">Goat Anti-Mouse IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>115005003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Mouse IgM&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>115005020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>112005167</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Hoechst 33342</td>
			<td>Life Technologies</td>
			<td>H3570</td>
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			<td height="21" style="height:21px;">Igepal</td>
			<td>Sigma Aldrich</td>
			<td>I3021-100ML&#160;</td>
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			<td height="21" style="height:21px;">Insulin&#160;</td>
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			<td>I6634&#160;</td>
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			<td>60615</td>
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			<td>Life Technologies</td>
			<td>23017015</td>
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			<td height="21" style="height:21px;">LB Medium</td>
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			<td>See stock solutions</td>
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			<td height="21" style="height:21px;">LB-Agar</td>
			<td></td>
			<td></td>
			<td>See stock solutions</td>
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			<td height="21" style="height:21px;">L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>C-7477</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Leibovitz&#39;s L-15 Medium</td>
			<td>Life Technologies</td>
			<td>11415064</td>
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		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G1626</td>
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			<td height="21" style="height:21px;">L-Glutamine- 200mM (100X) liquid</td>
			<td>Life Technologies</td>
			<td>25030081</td>
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		<tr height="21">
			<td height="21" style="height:21px;">LR Clonase II Plus enzyme</td>
			<td>Life Technologies</td>
			<td>12538-120</td>
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		<tr height="21">
			<td height="21" style="height:21px;">MEM, NEAA, no Glutamine</td>
			<td>Life Technologies</td>
			<td>10370088</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mouse &#945; &#946;III Tubulin&#160;</td>
			<td>Promega</td>
			<td>G7121</td>
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			<td height="21" style="height:21px;">Mouse &#945;MBP (monoclonal)</td>
			<td>Millipore</td>
			<td>MAB381</td>
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			<td height="21" style="height:21px;">Na pyruvate&#160;</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
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		<tr height="21">
			<td height="21" style="height:21px;">NAC</td>
			<td>Sigma Aldrich</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NcoI-HF</td>
			<td>NEB</td>
			<td>R3193S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEBuffer 4</td>
			<td>NEB</td>
			<td>B7004S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal medium</td>
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			<td>21103049</td>
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		<tr height="21">
			<td height="21" style="height:21px;">NGF (mouse)</td>
			<td>Alomone Labs</td>
			<td>N-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NT-3</td>
			<td>Peprotech</td>
			<td>&#160;450-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O1 antibody - Mouse anti-O1</td>
			<td>Millipore</td>
			<td>MAB344</td>
			<td>Alternative if O1 hybridoma cells are unavailable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O1 hybridoma cells</td>
			<td></td>
			<td></td>
			<td>Conditioned medium containing anti-O1 antibody to be used for immunopanning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O4 antibody - Mouse anti-O4</td>
			<td>Millipore</td>
			<td>MAB345</td>
			<td>Alternative if O4 hybridoma cells are unavailable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O4 hybridoma cells</td>
			<td></td>
			<td></td>
			<td>Conditioned medium containing anti-O4 antibody to be used for immunopanning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Competent Cells</td>
			<td>Life Technologies</td>
			<td>A10460</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">One Shot Stbl competent cells</td>
			<td>Life Technologies</td>
			<td>C7373-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Papain Suspension</td>
			<td>Worthington/Cooper</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">pBR8.91</td>
			<td></td>
			<td></td>
			<td>Kind gift from Dr Denham10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDGF-AA (Human)</td>
			<td>Peprotech</td>
			<td>PT10013A500&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin- Streptomycin 100X solution</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pENTRY4IRES2GFP</td>
			<td>Invitrogen</td>
			<td>11818-010&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylenimine (PEI)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>408727-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-ornithine&#160;</td>
			<td>Sigma Aldrich&#160;</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Progesterone&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease inhibitor tablet (Complete mini)</td>
			<td>Roche</td>
			<td>11836153001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td></td>
			<td></td>
			<td>Supplied with Clonase enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Putrescine</td>
			<td>Sigma Aldrich</td>
			<td>P-5780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit &#945; neurofilament</td>
			<td>Millipore</td>
			<td>AB1987&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit &#945;MBP (polyclonal)</td>
			<td>Millipore</td>
			<td>AB980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ran2 hybridoma cells</td>
			<td>ATCC</td>
			<td>TIB-119</td>
			<td>Conditioned medium containing anti-Ran2 antibody to be used for immunopanning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat anti CD140A/PDGFRa antibody</td>
			<td>BD Pharmingen</td>
			<td>558774</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SacII</td>
			<td>NEB</td>
			<td>R0157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SOC medium</td>
			<td></td>
			<td></td>
			<td>Supplied with competent bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium selenite&#160;</td>
			<td>Sigma Aldrich</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spe I</td>
			<td>NEB</td>
			<td>R0133S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA Ligase Buffer</td>
			<td>NEB</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE buffer pH8</td>
			<td></td>
			<td></td>
			<td>See stock solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TNE lysis buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trace Elements B</td>
			<td>Cellgro&#160;</td>
			<td>99-175-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transferrin (apo-Transferrin human)</td>
			<td>Sigma-Aldrich</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton&#160;X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T9201-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin Inhibitor From Chicken Egg White</td>
			<td>Roche</td>
			<td>10109878001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (1X), phenol red (0.05%)</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Unconjugated Griffonia Simplicifolia Lectin BSL-1</td>
			<td>Vector laboratories&#160;</td>
			<td>L-1100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3003-5G</td>
			<td></td>
		</tr>
	</tbody>
</table>

production and use of lentivirus to selectively transduce primary oligodendrocyte precursor cells for in vitro myelination assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

The flag-tagged extracellular signal-related kinase 1(Flag-Erk1) construct used for lentivirus production is verified by restriction enzyme digest of the constructs used, including both the 2K7 constructs and the packaging and accessory constructs required for virus production (Figure 3).
<img alt="Figure 3" src="/files/ftp_upload/52179/52179fig3highres.jpg" /> 
Figure 3: DNA construct verification. All DNA constructs...

Watch this Scientific Journal Video about Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays at JoVE.com

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

production-use-lentivirus-to-selectively-transduce-primary

Research

JoVE Journal

Developmental Biology

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...