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<img src="" 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="">Home</a></li> <li><a href="">About</a> <ul> <li><a href="">Abstract</a></li> <li><a href="">Goals</a></li>

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<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> <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>


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