Name: from mefs to matrigel i: passaging hescs in the presence of mefs
Uploaded: 2008-06-04T23:27:00
Description: Watch this Scientific Journal Video about From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs at JoVE.com

<p class="jove_content">Human embryonic stem cell studies in the Teitell lab are supported by a California Institute for Regenerative Medicine (CIRM) seed grant RS1-00313. We thank members of the Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, especially Dr. Amander Clark, Dr. Jerome Zack, and members of the UCLA Broad Institute Stem Cell Core Facility for their support of our studies.</p>

<p class="jove_title">Splitting human embryonic stem cells (hESCs) plated on mouse embryonic fibroblasts (MEFs)</p>
<p class="jove_content">Usually a confluent hESC plate can be split 1:6 to 1:10, depending on the particular hESC line. The split plate will become confluent again 5-7 days after splitting.</p>
<ol>
<li>Two days before splitting, gelatinize plates using a 0.1% gelatin solution. For a 6-well plate, add 2ml into each well and incubate plate in a 37&deg;C, 5% CO<sub>2</sub> tissue culture incubator overnight.</li>
<li>Plate MEFs on g...

<ol>
	<li>Thomson, J. a. m. e. s. A., Itskovitz-Eldor, J. o. s. e. p. h., Shapiro, S. a. n. d. e. r. S., Waknitz, M. i. c. h. e. l. l. e. A., Swiergiel, J. e. n. n. i. f. e. r. J., Marshall, V. i. v. i. e. n. n. e. S., Jones, J. e. f. f. r. e. y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Stem+Cell+Lines+Derived+from+Human+Blastocysts.">Embryonic Stem Cell Lines Derived from Human Blastocysts.</a> <em style='font-style: italic'>Science </em>. <b>282</b>, 1145-1147 (1998).</li><li>Xu, C. h. u. n. h. u. i., Inokuma, M. a. r. g. a. r. e. t. S., Denham, J. e. r. r. o. d., Golds, K. a. t. h. a. l. e. e. n., Kundu, P. r. a. t. i. m. a., Gold, J. o. s. e. p. h. D., Carpenter, M. e. l. i. s. s. a. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> <em style='font-style: italic'>Nature Biotechnology </em>. <b>19</b>, 971-974 (2001).</li></ol>

<p class="jove_content">This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. In the last step before plating, when the hESCs are resuspended, they should be of roughly uniform colony size and shape. Carefully pipette the hESC suspension up and down a few times to make the colonies smaller and more uniform, but not so much that single cells or very small colonies are generated.Immunofluorescence staining and microscopy or flow cytometry for hESC pluripotency markers, such as Oct...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Knockout Serum Replacer (KSR)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>DMEM/F12</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11330-057</td>
<td>&nbsp;</td>
<tr>
<td> Non-essential Amino Acids</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMax </td>
<td>Reagent</td>
<td>Gibco</td>
<td>35050-061</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11995-065</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>Reagent</td>
<td>Clontech</td>
<td>631107</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25030-081</td>
<td>&nbsp;</td>
<tr>
<td>BME</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP176-100</td>
<td>&nbsp;</td>
<tr>
<td>bFGF</td>
<td>Reagent</td>
<td>R&D Systems</td>
<td>233-FB-025</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV</td>
<td>Reagent</td>
<td>Gibco</td>
<td>17104-019</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>Reagent</td>
<td>Stem Cell Technologies</td>
<td>17105-041</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin / Streptomycin </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Gelatin</td>
<td>Reagent</td>
<td>Chemicon</td>
<td>ES-006-B</td>
<td>&nbsp;</td>
<tr>
<td>Matrigel</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>354277</td>
<td>&nbsp;</td>
<tr>
<td>Oct-4 antibody</td>
<td>Reagent</td>
<td>Santa Cruz Biotechnology</td>
<td>SC-9081</td>
<td>&nbsp;</td>
<tr>
<td>anti-h/mSSEA-4 Phycoerythrin Conjugated Mouse IgG3</td>
<td>Reagent</td>
<td>R&D Systems</td>
<td>FAB1435P</td>
<td>&nbsp;</td>
<tr>
<td>FITCI-conjugated antirabbit IgG</td>
<td>Reagent</td>
<td>Jackson ImmunoResearch Laboratories. Inc.</td>
<td>715-095-152</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
	

from mefs to matrigel i: passaging hescs in the presence of mefs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.


Watch this Scientific Journal Video about From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs at JoVE.com

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

<p class="jove_content">This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.</p>

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

Research

JoVE Journal

Biology

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generering av Myospheres Fra hESCs av Epigenetic Omprogrammering

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Isolering av Mitokondrier fra minimale mengder av Mouse Skeletal Muscle for høy gjennomstrømming Micro respirasjonsmålinger

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

Høy oppløsning Respirometry å måle mitokondrie funksjon av intakt Beta-cellene i nærvær av naturlige forbindelser

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

Measuring Crop Motility and Food Passaging in <em>Drosophila</em>

Measuring Crop Motility and Food Passaging in Drosophila

Måling av avling motilitet og mat passaging i <em>Drosophila</em>

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...