Team:Slovenia/Notebook

From 2012.igem.org

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<h3>Microscopy-alginate degradation (Microencapsulation)</h3>
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<h3>Microscopy-alginate beads degradation (Microencapsulation)</h3>
<p>
<p>
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To observe alginate beads degradation, 2000 kDa FITC-dexstran (Sigma) was added to 200 µL of culture medium containing alginate beads with immobilized HEK 293T cells. Because FITC-dexstran cannot penetrate into algnate beads, we can easily observe bead degradation uppon addition of alginate lyase from <i>Sphingobacterium multivorum</i> (Sigma).</p>
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To observe alginate beads degradation, 20 µL of 2000 kDa FITC-dexstran (1 mg/mL; Sigma) was added to 200 µL of culture medium containing alginate beads with immobilized HEK 293T cells. Because FITC-dexstran cannot penetrate into algnate beads, we can easily observe bead degradation uppon addition of alginate lyase from <i>Sphingobacterium multivorum</i> (Sigma).</p>
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<ol>
<li>Alginate beads suspended in culture medium were seeded into an 8-well microscope chamber (200 µL).</li>
<li>Alginate beads suspended in culture medium were seeded into an 8-well microscope chamber (200 µL).</li>
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<li>20 µL of 1 mg/mL FITC-dextran were added into well.</li>
<li>20 µL of 1 mg/mL FITC-dextran were added into well.</li>
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<li>After the dye has been evenly distributed throughout the suspension, 8 µL of Sphingobacterium multivorum alginate lyase were added.</li>
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<li>After the dye has been evenly distributed throughout the suspension, 8 µL of <i>Sphingobacterium multivorum</i> alginate lyase were added.</li>
<li>Microscope was set to capture images every 20 seconds.</li>
<li>Microscope was set to capture images every 20 seconds.</li>
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<li>Encapsulated cells were grown in DMEM culture medium supplemented with 10% FBS.</li>
<li>Encapsulated cells were grown in DMEM culture medium supplemented with 10% FBS.</li>
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<li>200 µL of microcapsule suspension were collected and alginate-PLL capsules were seeded into an 8-well microscope chamber.</li>
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<li>200 µL of microcapsule suspension was collected and alginate-PLL capsules were seeded into an 8-well microscope chamber.</li>
<li>5 µL of 7-AAD and 1 µL of Hoechst stain were added into well.</li>
<li>5 µL of 7-AAD and 1 µL of Hoechst stain were added into well.</li>

Revision as of 14:14, 26 September 2012