Difference between revisions of "Biomod/2013/OSU/protocol/MTT"

From OpenWetWare
Jump to: navigation, search
Line 79: Line 79:
  
 
</html>
 
</html>
[http://openwetware.org/index.php?title=Biomod/2013/OSU/results/protocol/MTT&action=edit Edit]
+
[http://openwetware.org/index.php?title=Biomod/2013/OSU/protocol/MTT&action=edit Edit]
  
 
{{OSUFooter}}
 
{{OSUFooter}}

Revision as of 15:11, 24 October 2013


<html> </div> </div> </div> <head> <style type='text/css'>

.firstHeading, #column-one, #p-bookmarks, #p-history , .portlet{ display:none; }

  1. bodyContent, #content{

display:none; background-color:#FFFFFF;

}

  1. column-content, .container, #globalWrapper, body{

background-color:#FFFFFF; }

p{ color:dimgrey; text-align:center; margin-left:auto; margin-right:auto; font-gamily:Calibri; font-size: 15px; }

.selflink{ display: table-cell;

  text-decoration: none;
  font-family: Gill Sans MT;
  outline: none;
  font-size: 20px;
  color: dimgrey;
  border-right: 2px solid dimgray;
  padding: 15px 25px; /* sets button size */
  font-weight:normal;

}

ul.navigation-bar {

  list-style-type: none;
  border: 2px solid dimgray;
  padding: 0px;
  margin-left: 20px;

width:615px;

  *line-height: 3.2em; /* IE6,IE7 */
  }

ul.navigation-bar li {

  display: table-cell;
  background-image: -webkit-linear-gradient(bottom, silver, azure);
  background-image:    -moz-linear-gradient(bottom, silver, azure);
  background-image:     -ms-linear-gradient(bottom, silver, azure);
  background-image:         linear-gradient(bottom, silver, azure);
  background-color: midnightblue; /* IE6...IE9 */
  *display: inline; /* IE6,IE7 */
  }


ul.navigation-bar li:hover {

  background-image: -webkit-linear-gradient(top, silver, azure);
  background-image:    -moz-linear-gradient(top, silver, azure);
  background-image:     -ms-linear-gradient(top, silver, azure);
  background-image:         linear-gradient(top, silver, azure);

}

ul.navigation-bar li a, ul.navigation-bar li ul li a {

  display: table-cell;
  text-decoration: none;
  font-family: Gill Sans MT;
  outline: none;
  font-size: 20px;
  color: dimgrey;
  border-right: 0px;
  padding: 15px 25px; /* sets button size */
  }

ul.navigation-bar li ul{ list-style: none; display: none; position: absolute; padding: 0px; margin: 1px 0px 0px -1px; background-color:#FFFFFF; border: 1px solid dimgrey; z-index:5; } ul.navigation-bar li ul li{ background-image: none; background-color:#FFFFFF; border-right: none; border: 0px solid dimgray; display: block; float: none; position: relative; }

ul.navigation-bar li ul li:hover{ background-image:none; background-color: lightgrey; }

ul.navigation-bar li:hover ul{ list-style: none; display: block; position: absolute; }

ul.navigation-bar li a{ display: block; }

.profile-right{ clear:both; }

.profile-right p{ text-align:left; float: left; } .profile-right img{ float: right; padding: 20px; }

.profile-left{ clear:both; }

.profile-left p{ text-align:left; float:left; }

.profile-left img{ float: left; padding:20px; }

  1. OSUfooter{

clear:both; }


  1. protocol-content table td{

border: 1px solid black; text-align:center; }

  1. protocol-content p{

text-align:left; }

.overlay-bg{ display:none; position:fixed; top:0; left:0; height:100%; width:100%; cursor: pointer; background: #000; background: rgba(0,0,0,0.75); }

.overlay-content{ background:#fff; padding:1%; width: 60%; position: relative; top:15%; left:50%; margin: 0 0 0 -30%; cursor: default; -webkit-border-radius: 5px; -moz-border-radius: 5px; border-radius: 5px; box-shadow: 0 0 5px rgba(0,0,0,0.9);

}

.close-btn{ float:right; font-size:x-large; margin-top:-10px; }

</style>


<script src="//ajax.googleapis.com/ajax/libs/jquery/1.10.1/jquery.min.js"></script>

<script type="text/javascript"> $(document).ready(function() {

 $('.show-popup').click(function(event){
 event.preventDefault();
 $('.overlay-bg').show();

});

$('.close-btn').click(function(){ $('.overlay-bg').hide(); });

$('.overlay-bg').click(function(){

 $('.overlay-bg').hide();

});

$('.overlay-content').click(function(){ return false; });

}); </script>

</head>

<!-- navbar -->

<a href="http://openwetware.org/wiki/Biomod/2013"> << Back to Team Pages</a>

<center>

<img src="http://openwetware.org/images/1/15/Ohiomod2013.jpg" height="60%" width="60%" style="margin-left:-40px;"> <div id="navbar" style="margin-left:auto; margin-right:auto;text-align:center;width:650px;height:auto;" > <ul id="homebutton" class="navigation-bar"> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU">Home</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/about">About</a> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/abstract">Abstract</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/goals">Goals</a></li>

<li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/about">Description</a> </ul> </li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/experiments">Project</a> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/experiments">Life of Project</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol">Protocols</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/labnotes">Lab Notes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/meetingnotes">Meeting Notes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/resources">Resources</a></li> </ul> </li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/results">Results</a> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/results">Experimental Results</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/data">Data</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/futurework">Future Work</a></li> </li> </ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/media">Media</a> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/projectvideo">Project Video</a><li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/animations">Animations</a><li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/videoblogs">Video Blog</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/photos">Photos</a></li> </ul> </li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/team">Team</a></li> </ul>

</div>

</center> </html>

<html> <center> <div id="protocol-content" style="width:65%;margin-left:auto;margin-right:auto;"> <h1>MTT Assay Protocol</h1> <ol> <li>Determine cell concentration using hemacytometer </li> <ol> <li>Take out cell line</li> <li>Remove specified amount from flask and transfer to 50 mL centrifuge tube</li> <li>Take 10 microliter sample and put into eppendorf tube</li> <li>Add 10 microliters of trypan blue to tube and mix</li> <li>Take 10 microliters and add to hemacytometer</li> <li>Count number of cells in each 4 x 4 grid, and divide by 4. Multiply by 2 (dilution factor)</li> </ol> The number obtained multiplied by 10^4 is the cell concentration <li>Put cells in each well of the plate at a concentration of 2.5*10^5 mL (50,000 per well)</li> <ol> <li>Centrifuge specified amount of cells in media (3 g 5 min 4 deg C)</li> <li>Decant and Wash with PBS</li> <li>Centrifuge and resuspend in media without serum</li> <li>Dilute concentration of cells to get specified concentration in each well</li> </ol>

<h2>Dilutions</h2> <h3>Do dilutions in sterile tubes inside of the cell hood</h3> <p>Dilute Daunorubicin (found in pink holder in door of fridge) to 20 micromolar using serum free media***Make twice as concentrated since it will be diluted by cells (20 micromolar=10 micromolar concentration, 10 micromolar=5 micromolar concentration, etc.) e.g. To make 1 mM add 20 microliters to 980 microliters media) <br> I recommend adding the media to all of the labeled tubes before beginning dilutions</p> <ol> <li value="5">Add 750 microliters of 20 um solution to 750 microliters media (5 µM label)</li> <li value="6">Add 600 microliters of 5 µM solution to 900 microliters of media in eppendorf tube (2 µM label)</li> <li value="7">Add 750 microliters of 2 µM to 750 microliters of media in eppendorf tube (1 µM label)</li> <li value="8">Add 750 microliters of 1 µM to 750 microliters of media in eppendorf tube (.5 µM label)</li> <li value="9">Add 300 microliters of .5 µM to 1200 microliters of media in eppendorf tube (.1 µM label)</li>

<li>Take loaded origami solution with specified concentration in PBS</li> <ol>

<li>Dilute with RPMI no serum Media to form 20 µM daunorubicin concentration (10 µM)</li>
<li>Use dilutions listed above to make remaining concentrations (5, 2, 1, .5, .1 µM)</li>
<li>Add media/origami solution to cells for a final volume of 200 microliters for each well (Triplicate for each concentration)</li>

</ol> <li>Incubate for 12 hours</li>


<p>After Incubation (Day One)</p> <ol> <li>Centrifuge cell plate (300 g 5 min 4 deg C)</li> <li>Aspirate media for each of the wells using Pasteur pipet</li> <li>Resuspend cells in PBS (200 microliter—use multichannel pipet)</li> <li>Centrifuge cell plate again (300 g 5 min 4 deg C)</li> <li>Resuspend cells in RPMI no serum media (200 microliter—use multichannel pipet)</li> <li>Put back in incubator for 24, 48 hours</li> </ol> <li>Performing MTT Assay (After 24,48 hour incubation)<li> <ol>

<li>Centrifuge cell plate ((300 g 5 min 4 deg C)</li> <li>Aspirate media</li> <li>Add 150 microliters of HL60 Media to each well</li> <li>Add 20 microliters of MTT solution (5 mg/mL of MTT salt in HL60 Media —make 25 microliters for each well which will be tested) to each well.</li> <li>Wrap with foil and put into incubator for approximately 2 hours</li> <ol> <li>Check after 2 hour to see if crystals are formed</li> <li>If a significant amount of crystals have formed take out of incubator</li> <li>If no crystals are formed wait longer (3-4 hours)</li> </ol> <p>Take out and aspirate media (be sure to not take up crystals)</p> <li>Resuspend in 200 microliters isopropanol</li> <li>Put on shaker plate for 10 mins until crystals are dissolved (or mix up and down with multichannel pipet, ensure that crystals dissolve and a purple color is formed in the wells)</li> <li>Read in plate reader at 560 nm and 670 nm wavelengths</li> </ol> <p>Note: Subtract 670 wavelength (background)</p>

</div> </center> </div>

</html> Edit


<html>

<head> <style>

  1. Footer table {
  width: 96.8%;
  max-width:2350px;
  bottom:0px;
  height:50px;   /* Height of the footer */
  font-weight:300;
  background-color: #2E2E2E;
   text-align:center;
   color: white;
   font-size:13px;
   border: 3px white solid; 

}

  1. Footer {
   clear: both; /*may be omitted*/
   position: absolute;
   bottom: 12px; 
   background-color:#fffff;
   width: 100%;
   height: 40px; /* or anything you like */
}


</style> </head> <div id="Footer" >

       <table>
           <tr>

<td id="osuicon" align="left"><a href="http://www.osu.edu" target="_blank"><img src="http://openwetware.org/images/c/cd/TheOhioStateUniversity-REV-Horiz-RGBHEX.png"height="30" width="190" /></td>

<td id="email">Contact us at <a href="mailto:theohiomod@gmail.com"><font color="white">theohiomod@gmail.com</a></td>

<td id="nblicon"><a href="http://mae.osu.edu/labs/nbl/"target="_blank"><img src="http://openwetware.org/images/e/e9/NBL_logo-RGBHEX.png"height="40" width="40"/></td>

<td id="fbicon"><a href="http://www.facebook.com/OhioMod?fref=photo"target="_blank"><img src="http://openwetware.org/images/3/3f/Facebook_icon.png"height="40" width="40"/></td>

           </tr>
       </table>
   </div>

</html>