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 macrophages 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> </p>
</td>
<td width="138">
<p> </p>
</td>
<td width="216">
<p> </p>
</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>MACROPHAGE DIFFERENTIATION base</strong></p>
<ol>
<li>500mL DMEM</li>
<li>50mL human AB serum (heat inactivated) – we test multiple vendors and multiple lots from each vendor and buy in bulk. The test is performed using healthy human donor peripheral blood monocytes differentiated in MACROPHAGE DIFFERENTIATION complete medium with different human sera lots. This is critically important as we have found massive variability in the capacity of human sera to support macrophage differentiation. A “good” serum should result in full conversion of monocytes into fried egg appearing macrophages within 7 days.</li>
<li>5mL GlutaMAX</li>
<li>Pen/Strep</li>
</ol>
<p> </p>
<p><strong>MACROPHAGE DIFFERENTIATION complete</strong></p>
<ol>
<li>To a 50mL aliquot of the base medium, add 1 ng/mL M-CSF (=5 ul of 10 μg/mL stock solution)</li>
</ol>
<p> </p>

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