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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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<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>

<p style="font-size:140%; color:black;">Dilutions</p> <p style="font-size:120%; color:black;">Do dilutions in sterile tubes inside of the cell hood</p> <p style="color:black;">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> </ol> <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 style="font-size:120%;color:black;">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> <li>Take out and aspirate media (be sure to not take up crystals)</li> <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 style="color:black;">Note: Subtract 670 wavelength (background)</p>



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