Biomod/2013/OSU/protocol/MTT

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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/drugloading">
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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>
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<li>Performing MTT Assay (After 24,48 hour incubation)<li>
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<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>
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<p style="color:black;">Take out and aspirate media (be sure to not take up crystals)</p>
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<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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MTT Assay Protocol

  1. Determine cell concentration using hemacytometer
    1. Take out cell line
    2. Remove specified amount from flask and transfer to 50 mL centrifuge tube
    3. Take 10 microliter sample and put into eppendorf tube
    4. Add 10 microliters of trypan blue to tube and mix
    5. Take 10 microliters and add to hemacytometer
    6. Count number of cells in each 4 x 4 grid, and divide by 4. Multiply by 2 (dilution factor)
    The number obtained multiplied by 10^4 is the cell concentration
  2. Put cells in each well of the plate at a concentration of 2.5*10^5 mL (50,000 per well)
    1. Centrifuge specified amount of cells in media (3 g 5 min 4 deg C)
    2. Decant and Wash with PBS
    3. Centrifuge and resuspend in media without serum
    4. Dilute concentration of cells to get specified concentration in each well

    Dilutions

    Do dilutions in sterile tubes inside of the cell hood

    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)
    I recommend adding the media to all of the labeled tubes before beginning dilutions

    1. Add 750 microliters of 20 um solution to 750 microliters media (5 µM label)
    2. Add 600 microliters of 5 µM solution to 900 microliters of media in eppendorf tube (2 µM label)
    3. Add 750 microliters of 2 µM to 750 microliters of media in eppendorf tube (1 µM label)
    4. Add 750 microliters of 1 µM to 750 microliters of media in eppendorf tube (.5 µM label)
    5. Add 300 microliters of .5 µM to 1200 microliters of media in eppendorf tube (.1 µM label)
  3. Take loaded origami solution with specified concentration in PBS
    1. Dilute with RPMI no serum Media to form 20 µM daunorubicin concentration (10 µM)
    2. Use dilutions listed above to make remaining concentrations (5, 2, 1, .5, .1 µM)
    3. Add media/origami solution to cells for a final volume of 200 microliters for each well (Triplicate for each concentration)
  4. Incubate for 12 hours
  5. After Incubation (Day One)

    1. Centrifuge cell plate (300 g 5 min 4 deg C)
    2. Aspirate media for each of the wells using Pasteur pipet
    3. Resuspend cells in PBS (200 microliter—use multichannel pipet)
    4. Centrifuge cell plate again (300 g 5 min 4 deg C)
    5. Resuspend cells in RPMI no serum media (200 microliter—use multichannel pipet)
    6. Put back in incubator for 24, 48 hours
  6. Performing MTT Assay (After 24,48 hour incubation)
    1. Centrifuge cell plate ((300 g 5 min 4 deg C)
    2. Aspirate media
    3. Add 150 microliters of HL60 Media to each well
    4. 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.
    5. Wrap with foil and put into incubator for approximately 2 hours
      1. Check after 2 hour to see if crystals are formed
      2. If a significant amount of crystals have formed take out of incubator
      3. If no crystals are formed wait longer (3-4 hours)
    6. Take out and aspirate media (be sure to not take up crystals)
    7. Resuspend in 200 microliters isopropanol
    8. 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)
    9. Read in plate reader at 560 nm and 670 nm wavelengths

    Note: Subtract 670 wavelength (background)


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