Biomod/2013/OSU/protocol/MTT: Difference between revisions
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<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/folding_reaction"> | |||
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<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/gel_prep"> | |||
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<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/negative_staining"> | |||
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<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/custom_tem_grids"> | |||
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<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/drugloading"> | |||
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<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/TIRF"> | |||
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<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/MTT"> | |||
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<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/cellanalysis"> | |||
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<li>Put back in incubator for 24, 48 hours</li> | <li>Put back in incubator for 24, 48 hours</li> | ||
</ol> | </ol> | ||
<li>Performing MTT Assay (After 24,48 hour incubation)<li> | <li>Performing MTT Assay (After 24,48 hour incubation)</li> | ||
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<li>If no crystals are formed wait longer (3-4 hours)</li> | <li>If no crystals are formed wait longer (3-4 hours)</li> | ||
</ol> | </ol> | ||
< | <li>Take out and aspirate media (be sure to not take up crystals)</li> | ||
<li>Resuspend in 200 microliters isopropanol</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>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> | ||
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<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>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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