Difference between revisions of "Team:TecCEM/Experiments"

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             <div class="row">
 
             <div class="row">
 
                 <div class="col">
 
                 <div class="col">
                     <p>This is our experiment section. Here we compile important protocols for the development of TecTissue, ranging from our bacterial transformation procedures to our cell proliferation assays.  
+
                     <p>This is our experiment section. Here we compile important protocols for the development of
We also address cell culture maintenance and protein loaded chitosan nanoparticles.  
+
                        TecTissue, ranging from our bacterial transformation procedures to our cell proliferation
Here you may find the protocol for our growth factor delivery to damaged cells and how much harm can be inflicted in vitro.
+
                        assays.
 +
                        We also address cell culture maintenance and protein loaded chitosan nanoparticles.
 +
                        Here you may find the protocol for our growth factor delivery to damaged cells and how much
 +
                        harm can be inflicted in vitro.
 
                     </p>
 
                     </p>
 
                 </div>
 
                 </div>
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         </div>
 
         </div>
 
         <div class="container mt-3">
 
         <div class="container mt-3">
             <div class="row">
+
            <h1>Protocols</h1>
 +
            <h2>Chitosan nanoparticles</h2>
 +
             <div class="row" id="encapsulation">
 +
                <div class="col">
 +
                    <h3>Protein encapsulation protocol</h3>
 +
                    <div class="mb-3">
 +
                        <h4>Reactants</h4>
 +
                        <ul>
 +
                            <li>Chitosan low molecular weight from Sigma-Aldrich</li>
 +
                            <li>TPP from Sigma-Aldrich</li>
 +
                            <li>NaOH 1M</li>
 +
                            <li>Acetic acid 1M</li>
 +
                            <li>Distilled water</li>
 +
                            <li>Protein of interest (10 mg/mL)</li>
 +
                        </ul>
 +
                    </div>
 +
                    <h4>Procedure</h4>
 +
                    <div class="mb-3">
 +
                        <em>
 +
                            <h5>Stock solutions</h5>
 +
                        </em>
 +
                        <ol>
 +
                            <li>In a 15 mL Falcon tube add 30 mg of chitosan and 10 mL of distilled water (to get a
 +
                                solution with a concentration of 3 mg/mL).</li>
 +
                            <li>Add 10 microliters of acetic acid for each mL of chitosan solution to solubilize the
 +
                                chitosan.
 +
                                To adjust the pH acetic acid and NaOH should be used.</li>
 +
                            NOTE: the pH should be adjusted depending on your protein of interest, taking into account
 +
                            the isoelectric point, always maintaining the chitosan solution positively charged (pH &lt;
 +
                            6.5)
 +
                            and the protein of interest negatively charged (preferred).
 +
                            <li> In another 15 mL falcon
 +
                                tube add 10 mg of TPP and 10 mL of distilled water (to get a concentration of 1 mg/mL).</li>
 +
                        </ol>
 +
                    </div>
 +
                    <div class="mb-3">
 +
                        <em>
 +
                            <h5>Nanoparticle preparation</h5>
 +
                        </em>
 +
                        <ol>
 +
                            <li>In a 20 mL beaker add 1 mL of chitosan solution and 100 uL of your protein, stir the
 +
                                mix at 1100 rpm with a magnetic stirrer (the size of nanoparticles is affected by rpm
 +
                                value; for smaller nanoparticles use higher rpm).</li>
 +
                            <li>Take 1 mL of the TPP solution and add it to the mix dropwise.</li>
 +
                            <li>Continue stirring for 1 hour.</li>
 +
                        </ol>
 +
                    </div>
 +
                    <div class="mb-3">
 +
                        <em>
 +
                            <h5>Particle collection</h5>
 +
                        </em>
 +
                        <ol>
 +
                            <li>Transfer the mix to 2 1.5 mL Eppendorf tubes.</li>
 +
                            <em>NOTE: If nanoparticles are to be extracted centrifuge the tubes at 20,000 rpm for 30
 +
                                minutes at 4°C.</em>
 +
                            <li>Eliminate the supernatant.</li>
 +
                            <li>The pellet will contain your protein of interest.</li>
 +
                            <li>If nanoparticles are to be used for liberation measurements or suspended in a
 +
                                controlled pH solution, resuspend well and store at 4 °C.</li>
 +
                        </ol>
 +
                    </div>
 +
                    <div class="mb-3">
 +
                        <em>
 +
                            <h5>TEM preparation</h5>
 +
                        </em>
 +
                        <em>To visualize chitosan nanoparticles some previous preparation steps must be carried out
 +
                            (this preparation protocol may vary).</em>
 +
                        <ol>
 +
                            <li>A film of Formvar has to be previously prepared and used to coat a glass slide for the
 +
                                creation of an 80-120 μm thick membrane.</li>
 +
                            <li>Place a copper grid on the Formvar membrane for it to be absorbed and later removed
 +
                                with a needle.</li>
 +
                            <li>Add 20 μL of your solution of interest into the grid and let it be absorbed. Add a
 +
                                solution of 1% (w/v) phosphotungstic acid until the sample dries.</li>
 +
                            <li>View in a transmission electron microscope.</li>
 +
                            <em>NOTE: Samples were observed at 150,000x.</em>
 +
                        </ol>
 +
                    </div>
 +
                </div>
 +
            </div>
 +
            <div class="row" id="encapsulation-efficiency">
 +
                <div class="col">
 +
                    <h3>Protein encapsulation efficiency protocol</h3>
 +
                </div>
 +
            </div>
 +
            <div class="row" id="liberation-and-stability">
 
                 <div class="col">
 
                 <div class="col">
                    <h1>Protocols</h1>
 
                    <h2>Chitosan nanoparticles</h2>
 
                    <h3 id="encapsulation">Protein encapsulation protocol</h3>
 
                    <p>
 
                        <b>Reactants</b>&nbsp;
 
<ul>
 
<li>Chitosan low molecular weight from Sigma-Aldrich</li>
 
<li>TPP from Sigma-Aldrich</li>
 
<li>NaOH 1M</li>
 
<li>Acetic acid 1M</li>
 
<li>Distilled water</li>
 
<li>Protein of interest (10 mg/mL)</li>
 
</ul>
 
  
<p>&nbsp;&nbsp;</p>
+
                    <h3>Protein liberation and stability protocol</h3>
<b>Procedure</b>
+
<p></p>
+
<i>Stock solutions</i><p>&nbsp;</p>
+
<ol>
+
<li>In a 15 mL Falcon tube add 30 mg of chitosan and 10 mL of distilled water (to get a solution with a concentration of 3 mg/mL).</li>
+
<li>Add 10 microliters of acetic acid for each mL of chitosan solution to solubilize the chitosan.
+
To adjust the pH acetic acid and NaOH should be used.</li>
+
NOTE: the pH should be adjusted depending on your protein of interest, taking into account the isoelectric point, always maintaining the chitosan solution positively charged (pH<6.5) and the protein of interest negatively charged (preferred).
+
<li>In another 15 mL falcon tube add 10 mg of TPP and 10 mL of distilled water (to get a concentration of 1 mg/mL).</li>
+
</ol>
+
<p>&nbsp;</p>
+
<i>Nanoparticle preparation</i><p></p>
+
<ol>
+
<li>In a 20 mL beaker add 1 mL of chitosan solution and 100 uL of your protein, stir the mix at 1100 rpm with a magnetic stirrer (the size of nanoparticles is affected by rpm value; for smaller nanoparticles use higher rpm).</li>
+
<li>Take 1 mL of the TPP solution and add it to the mix dropwise.</li>
+
<li>Continue stirring for 1 hour.</li>
+
</ol>
+
<p>&nbsp;</p>
+
 
+
<i>Particle collection</i>&nbsp; <p></p>
+
<ol>
+
<li>Transfer the mix to 2 1.5 mL Eppendorf tubes.</li>
+
NOTE: If nanoparticles are to be extracted centrifuge the tubes at 20,000 rpm for 30 minutes at 4°C.
+
<li>Eliminate the supernatant.</li>
+
<li>The pellet will contain your protein of interest.</li>
+
<li>If nanoparticles are to be used for liberation measurements or suspended in a controlled pH solution, resuspend well and store at 4 °C.</li></ol>
+
<p>&nbsp;</p>
+
 
+
<i>TEM preparation</i><p></p>
+
 
+
<i>To visualize chitosan nanoparticles some previous preparation steps must be carried out (this preparation protocol may vary).</i>
+
 
+
<ol>
+
<li>A film of Formvar has to be previously prepared and used to coat a glass slide for the  creation of an 80-120 μm thick membrane.</li>
+
<li>Place a copper grid on the Formvar membrane for it to be absorbed and later removed with a needle.</li>
+
<li>Add 20 μL of your solution of interest into the grid and let it be absorbed. Add a solution of 1% (w/v) phosphotungstic acid until the sample dries.</li>
+
<li>View in a transmission electron microscope.</li>
+
<p>&nbsp;</p>
+
NOTE: Samples were observed at 150,000x.
+
 
+
                    </p>
+
 
                 </div>
 
                 </div>
 
             </div>
 
             </div>

Revision as of 07:57, 17 October 2018

Cell Gif

Experiments

This is our experiment section. Here we compile important protocols for the development of TecTissue, ranging from our bacterial transformation procedures to our cell proliferation assays. We also address cell culture maintenance and protein loaded chitosan nanoparticles. Here you may find the protocol for our growth factor delivery to damaged cells and how much harm can be inflicted in vitro.

Protocols

Chitosan nanoparticles

Protein encapsulation protocol

Reactants

  • Chitosan low molecular weight from Sigma-Aldrich
  • TPP from Sigma-Aldrich
  • NaOH 1M
  • Acetic acid 1M
  • Distilled water
  • Protein of interest (10 mg/mL)

Procedure

Stock solutions
  1. In a 15 mL Falcon tube add 30 mg of chitosan and 10 mL of distilled water (to get a solution with a concentration of 3 mg/mL).
  2. Add 10 microliters of acetic acid for each mL of chitosan solution to solubilize the chitosan. To adjust the pH acetic acid and NaOH should be used.
  3. NOTE: the pH should be adjusted depending on your protein of interest, taking into account the isoelectric point, always maintaining the chitosan solution positively charged (pH < 6.5) and the protein of interest negatively charged (preferred).
  4. In another 15 mL falcon tube add 10 mg of TPP and 10 mL of distilled water (to get a concentration of 1 mg/mL).
Nanoparticle preparation
  1. In a 20 mL beaker add 1 mL of chitosan solution and 100 uL of your protein, stir the mix at 1100 rpm with a magnetic stirrer (the size of nanoparticles is affected by rpm value; for smaller nanoparticles use higher rpm).
  2. Take 1 mL of the TPP solution and add it to the mix dropwise.
  3. Continue stirring for 1 hour.
Particle collection
  1. Transfer the mix to 2 1.5 mL Eppendorf tubes.
  2. NOTE: If nanoparticles are to be extracted centrifuge the tubes at 20,000 rpm for 30 minutes at 4°C.
  3. Eliminate the supernatant.
  4. The pellet will contain your protein of interest.
  5. If nanoparticles are to be used for liberation measurements or suspended in a controlled pH solution, resuspend well and store at 4 °C.
TEM preparation
To visualize chitosan nanoparticles some previous preparation steps must be carried out (this preparation protocol may vary).
  1. A film of Formvar has to be previously prepared and used to coat a glass slide for the creation of an 80-120 μm thick membrane.
  2. Place a copper grid on the Formvar membrane for it to be absorbed and later removed with a needle.
  3. Add 20 μL of your solution of interest into the grid and let it be absorbed. Add a solution of 1% (w/v) phosphotungstic acid until the sample dries.
  4. View in a transmission electron microscope.
  5. NOTE: Samples were observed at 150,000x.

Protein encapsulation efficiency protocol

Protein liberation and stability protocol