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

From OpenWetWare
Jump to: navigation, search
Line 170: Line 170:
[http://openwetware.org/index.php?title=Biomod/2013/OSU/protocol/TIRF&action=edit Edit]

Latest revision as of 06:29, 26 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 */


ul.navigation-bar {

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


  *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; }


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

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



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




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

}); </script>


<!-- navbar -->

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


<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>


</center> </html>


<center> <table id="Table_01" width="801" height="126" border="0" cellpadding="0" cellspacing="0"> <tr> <td rowspan="4" align="left" valign="top">

                       <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/folding_reaction">

<img src="http://openwetware.org/images/9/9e/Protocol_01.gif" width="129" height="125" alt=""></a></td> <td rowspan="2" align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/gel_prep"> <img src="http://openwetware.org/images/4/48/Protocol_02.gif" width="99" height="123" border="0" alt=""></a></td> <td align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/negative_staining"> <img src="http://openwetware.org/images/e/ed/Protocol_03.gif" width="116" height="122" border="0" alt=""></a></td> <td rowspan="2" align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/custom_tem_grids"> <img src="http://openwetware.org/images/a/ae/Protocol_04.gif" width="93" height="123" border="0" alt=""></a></td> <td colspan="2" rowspan="2" align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/drugloading"> <img src="http://openwetware.org/images/e/e9/Protocol_05.gif" width="132" height="123" border="0" alt=""></a></td> <td colspan="2" rowspan="2" align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/TIRF"> <img src="http://openwetware.org/images/f/fd/Protocol_06.gif" width="137" height="123" border="0" alt=""></a></td> <td rowspan="2" align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/MTT"> <img src="http://openwetware.org/images/2/20/Protocol_07.gif" width="94" height="123" border="0" alt=""></a></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="1" height="122" alt=""></td> </tr> <tr> <td rowspan="3"> <img src="http://openwetware.org/images/0/0b/Protocol_08.gif" width="116" height="3" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="1" height="1" alt=""></td> </tr> <tr> <td rowspan="2"> <img src="http://openwetware.org/images/5/59/Protocol_09.gif" width="99" height="2" alt=""></td> <td colspan="2" rowspan="2"> <img src="http://openwetware.org/images/e/e4/Protocol_10.gif" width="211" height="2" alt=""></td> <td colspan="2" align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/cellanalysis"> <img src="http://openwetware.org/images/3/3c/Protocol_11.gif" width="108" height="1" border="0" alt=""></a></td> <td colspan="2" rowspan="2"> <img src="http://openwetware.org/images/8/83/Protocol_12.gif" width="137" height="2" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="1" height="1" alt=""></td> </tr> <tr> <td colspan="2"> <img src="http://openwetware.org/images/7/77/Protocol_13.gif" width="108" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="1" height="1" alt=""></td> </tr> <tr> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="129" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="99" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="116" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="93" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="118" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="14" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="94" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="43" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="94" height="1" alt=""></td> <td></td> </tr> </table> </center>

<div id="protocol-content" style="width:65%;margin-left:auto;margin-right:auto;">

<h1>TIRF protocol (Seeding plate)</h1> <ol> <li>Determine cell concentration using hemacytometer </li> <ol> <li>Take out cell line</li> <li>Remove specified amount from flask (start with 10 mL) 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> <p style="color:black;">The number obtained multiplied by 10^4 is the cell concentration/mL</p> </ol> <li>Put cells in each well of a 24 hour well plate at 200,000 cells per well</li> <ol> <li>Centrifuge specified amount of cells in media (300 g 5 min 4 deg C)</li> <li>Decant and Wash with PBS</li> <li>Centrifuge and resuspend in media without serum (300 g 5 min 4 deg C)</li> <li>Add amount of specified media with cells to obtain desired 200,000 cells per well (add 200 microliters of cells at concentration of 1 million per mL)</li> </ol>

<p style="color:black;"><b>Dilutions</b></p> <p style="color:black;">Do dilutions in sterile tubes inside of the cell hood Dilute Daunorubicin (found in pink holder in door of fridge) to 20 micromolar ***Make twice as concentrated since it will be diluted by cells (20 micromolar=10 micromolar concentration, 10 micromolar=5 micromolar concentration, etc.) e.g. 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>Add 500 microliters of 20 um solution to 500 microliters media (5 µM label)</li> <li>Add 400 microliters of 5 µM solution to 600 microliters of media in eppendorf tube (2 µM label)</li> <li>Add 500 microliters of 2 µM to 500 microliters of media in eppendorf tube (1 µM label)</li> <li>Add 500 microliters of 1 µM to 500 microliters of media in eppendorf tube (.5 µM label)</li> <li>Add 200 microliters of .5 µM to 800 microliters of media in eppendorf tube (.1 µM label)</li> </ol>

<li>Add Daunorubicin solution to cell plate wells</li> <ol> <li>Add 200 microliters of each concentration to specified cell plate wells</li> <li>Put into incubator (label with name/date/what is in each well)</li> </ol>

<li>Incubate for 12 hours</li>

<p style="color:black;">Sample Plate Layout</p> <table> <tr> <td colspan="4">Cell Plate Layout</td> </tr> <tr> <td width="25%">10 uM Dauno</td><td width="25%">5 uM Dauno</td><td width="25%">2 uM Dauno</td><td width="25%">Cells only</td> </tr> <tr> <td>1 uM Dauno</td><td width="25%">.5 uM Dauno</td><td width="25%">.1 uM Dauno</td><td width="25%">Cells only</td> </tr> </table>

<p style="color:black;">After Incubation (Day One)</p> <ol> <li>1. Take out samples and into labeled sterile eppendorf tubes (make sure to mix up and down a few times before putting in eppendorf tubes)</li> <li>Centrifuge (3 g 5 min 4 deg C)</li> <li>Decant media, Resuspend cells in PBS (400 microliter)</li> <li>Centrifuge (3 g 5 min 4 deg C)</li> <li>Decant, Resuspend cells in HL media with serum (400 microliter)</li> <li>Add the samples to a new labeled 24 well plate</li> <li>Put back in incubator for 24, 48 hours</li> </ol>

<p style="color:black;">Day of Imaging</p> <ol> <li>Add 100 microliters poly L-lysine to imaging plate (8 well imaging plate)</li> <li>Aspirate/take out cells from culture plate and add to eppendorf tube</li> <li>Centrifuge (3 g 5 min 4 deg C)</li> <li>Decant Media. Resuspend in 200 microliters PBS</li> <li>Centrifuge (3 g 5 min 4 deg C)</li> <li>Decant PBS. Resuspend in 200 microliters RPMI 1640 clear media </li> <li>Take off poly L-lysine from imaging plate</li> <li>Wash imaging plate wells with 200 microliter PBS</li> <li>Add 2 microliters 7 A.A.D. to cells in clear media. </li> <li>Mix and add to imaging plate</li> </ol>


</div> </div>



<head> <style>

  1. Footer table {
  width: 96.8%;
  height:50px;   /* Height of the footer */
  background-color: #2E2E2E;
   color: white;
   border: 3px white solid; 


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

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


<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>