Biomod/2013/OSU/protocol/TIRF

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TIRF protocol (Seeding plate)

  1. Determine cell concentration using hemacytometer
    1. Take out cell line
    2. Remove specified amount from flask (start with 10 mL) 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)
    7. The number obtained multiplied by 10^4 is the cell concentration/mL

  2. Put cells in each well of a 24 hour well plate at 200,000 cells per well
    1. Centrifuge specified amount of cells in media (300 g 5 min 4 deg C)
    2. Decant and Wash with PBS
    3. Centrifuge and resuspend in media without serum (300 g 5 min 4 deg C)
    4. 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)

    Dilutions

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

    1. Add 500 microliters of 20 um solution to 500 microliters media (5 µM label)
    2. Add 400 microliters of 5 µM solution to 600 microliters of media in eppendorf tube (2 µM label)
    3. Add 500 microliters of 2 µM to 500 microliters of media in eppendorf tube (1 µM label)
    4. Add 500 microliters of 1 µM to 500 microliters of media in eppendorf tube (.5 µM label)
    5. Add 200 microliters of .5 µM to 800 microliters of media in eppendorf tube (.1 µM label)
  3. Add Daunorubicin solution to cell plate wells
    1. Add 200 microliters of each concentration to specified cell plate wells
    2. Put into incubator (label with name/date/what is in each well)
  4. Incubate for 12 hours
  5. Sample Plate Layout

    Cell Plate Layout
    10 uM Dauno5 uM Dauno2 uM DaunoCells only
    1 uM Dauno.5 uM Dauno.1 uM DaunoCells only

    After Incubation (Day One)

    1. 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)
    2. Centrifuge (3 g 5 min 4 deg C)
    3. Decant media, Resuspend cells in PBS (400 microliter)
    4. Centrifuge (3 g 5 min 4 deg C)
    5. Decant, Resuspend cells in HL media with serum (400 microliter)
    6. Add the samples to a new labeled 24 well plate
    7. Put back in incubator for 24, 48 hours

    Day of Imaging

    1. Add 100 microliters poly L-lysine to imaging plate (8 well imaging plate)
    2. Aspirate/take out cells from culture plate and add to eppendorf tube
    3. Centrifuge (3 g 5 min 4 deg C)
    4. Decant Media. Resuspend in 200 microliters PBS
    5. Centrifuge (3 g 5 min 4 deg C)
    6. Decant PBS. Resuspend in 200 microliters RPMI 1640 clear media
    7. Take off poly L-lysine from imaging plate
    8. Wash imaging plate wells with 200 microliter PBS
    9. Add 2 microliters 7 A.A.D. to cells in clear media.
    10. Mix and add to imaging plate
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