TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is a modification of a previously published method by Wilgenburg et al (<a href="https://www.ncbi.nlm.nih.gov/pubmed/23951090" target="_blank">PLoS One, 2013</a>) to obtain monocytes and Dendritic cells (DCs) from induced Pluripotent Stem Cells (iPSCs) lines. </p>

[BEFORE_START]
<p><strong>1. Order neccessary supplies</strong></p>
<p> </p>
<table>
<tbody>
<tr>
<td width="175">
<p><strong>Item</strong></p>
</td>
<td width="138">
<p><strong>Catalog #</strong></p>
</td>
<td width="216">
<p><strong>Vendor</strong></p>
</td>
</tr>
<tr>
<td colspan="3" width="529">
<p><strong>hES Medium</strong></p>
</td>
</tr>
<tr>
<td width="175">
<p>KO DMEM</p>
</td>
<td width="138">
<p>10829-018</p>
</td>
<td width="216">
<p>Gibco</p>
</td>
</tr>
<tr>
<td width="175">
<p>KO Serum replacement</p>
</td>
<td width="138">
<p>10828</p>
</td>
<td width="216">
<p>Gibco</p>
</td>
</tr>
<tr>
<td width="175">
<p>NE Amino Acids</p>
</td>
<td width="138">
<p>M11-003</p>
</td>
<td width="216">
<p>PAA</p>
</td>
</tr>
<tr>
<td width="175">
<p>GlutaMAX</p>
</td>
<td width="138">
<p>35050</p>
</td>
<td width="216">
<p>Gibco</p>
</td>
</tr>
<tr>
<td width="175">
<p>2-Mercaptoethanol</p>
</td>
<td width="138">
<p>31350-010</p>
</td>
<td width="216">
<p>Gibco</p>
</td>
</tr>
<tr>
<td width="175">
<p>bFGF</p>
</td>
<td width="138">
<p>4114-TC-01M</p>
</td>
<td width="216">
<p>&amp;D Systems</p>
</td>
</tr>
<tr>
<td width="175">
<p>Pen/Strep</p>
</td>
<td width="138">
<p>P11-010</p>
</td>
<td width="216">
<p>PAA</p>
</td>
</tr>
<tr>
<td width="175">
<p> </p>
</td>
<td width="138">
<p> </p>
</td>
<td width="216">
<p> </p>
</td>
</tr>
<tr>
<td colspan="3" width="529">
<p><strong>X-VIVO FACTORY base</strong></p>
</td>
</tr>
<tr>
<td width="175">
<p>X-VIVO 15</p>
</td>
<td width="138">
<p>BE04-418</p>
</td>
<td width="216">
<p>Lonza</p>
</td>
</tr>
<tr>
<td width="175">
<p>GlutaMAX</p>
</td>
<td width="138">
<p>35050</p>
</td>
<td width="216">
<p>Gibco</p>
</td>
</tr>
<tr>
<td width="175">
<p>2-Mercaptoethanol</p>
</td>
<td width="138">
<p>31350-010</p>
</td>
<td width="216">
<p>Gibco</p>
</td>
</tr>
<tr>
<td width="175">
<p> </p>
</td>
<td width="138">
<p> </p>
</td>
<td width="216">
<p> </p>
</td>
</tr>
<tr>
<td colspan="3" width="529">
<p><strong>Additional Growth Factors</strong></p>
</td>
</tr>
<tr>
<td width="175">
<p>M-CSF</p>
</td>
<td width="138">
<p>300-25</p>
</td>
<td width="216">
<p>PeproTech</p>
</td>
</tr>
<tr>
<td width="175">
<p>IL-3</p>
</td>
<td width="138">
<p>200-03</p>
</td>
<td width="216">
<p>PeproTech</p>
</td>
</tr>
<tr>
<td width="175">
<p>Rock inhibitor: Y-27632 dihydrochloride</p>
</td>
<td width="138">
<p>1254</p>
</td>
<td width="216">
<p>Tocris</p>
</td>
</tr>
<tr>
<td width="175">
<p>GM-CSF</p>
</td>
<td width="138">
<p> 300-03</p>
</td>
<td width="216">
<p>PeproTech</p>
</td>
</tr>
<tr>
<td width="175">
<p>IL-4</p>
</td>
<td width="138">
<p>200-04</p>
</td>
<td width="216">
<p>PeproTech</p>
</td>
</tr>
<tr>
<td width="175"> </td>
<td width="138"> </td>
<td width="216"> </td>
</tr>
<tr>
<td colspan="3" width="529">
<p><strong>Other</strong></p>
</td>
</tr>
<tr>
<td width="175">
<p>Ultra-low attachment 6 well plates</p>
</td>
<td width="138">
<p>3471</p>
</td>
<td width="216">
<p>Corning  from Appleton Woods/or Fisher Scientific Ltd</p>
</td>
</tr>
<tr>
<td width="175">
<p>hESC-Qualified Matrix Matrigel</p>
</td>
<td width="138">
<p>354277</p>
</td>
<td width="216">
<p>Corning</p>
</td>
</tr>
<tr>
<td width="175">
<p>mTeSR1 media</p>
</td>
<td width="138">
<p>05850</p>
</td>
<td width="216">
<p>Stem Cell Technologies</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>2. Prepare solutions</strong></p>
<p> </p>
<p><strong>X-VIVO Factory base medium </strong></p>
<ol>
<li>500mL X-VIVO 15</li>
<li>5mL Glutamax (100X)</li>
<li>500μL 2-ME (1000x)</li>
</ol>
<p> </p>
<p><strong>FACTORY medium complete</strong></p>
<ol>
<li>25 mL of basic medium</li>
<li>250 μL MCSF / IL-3 cocktail</li>
</ol>
<p>            Stock MCSF = 20 μg/mL (final = 200 ng/mL)</p>
<p>            Stock IL-3 = 5 μg/mL (final = 50 ng/mL)</p>
<ol start="3">
<li>Pen/Strep</li>
</ol>
<p> </p>
<p><strong>DC DIFFERENTIATION base</strong></p>
<ol>
<li>500 ml CellGenix GMP DC Medium (20801-0500) [Serum Free]</li>
</ol>
<p><strong>DC DIFFERENTIATION complete</strong></p>
<ol>
<li>To a 15 mL aliquot of the base medium, add 50 ng/ml IL-4 and 50 ng/ml GM-CSF (150 ul of alliquotted and mixed cytokines each at 100X [5 ug/ml]) [Do not freeze thaw cytokines. Prepare single use aliquots in CellGenix GMP DC medium and store at -80C]</li>
</ol>
<p> </p>

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