Biomod/2013/OSU/protocol/TIRF
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TIRF protocol (Seeding plate)
- Determine cell concentration using hemacytometer
- Take out cell line
- Remove specified amount from flask (start with 10 mL) and transfer to 50 mL centrifuge tube
- Take 10 microliter sample and put into eppendorf tube
- Add 10 microliters of trypan blue to tube and mix
- Take 10 microliters and add to hemacytometer
- Count number of cells in each 4 x 4 grid, and divide by 4. Multiply by 2 (dilution factor)
- Put cells in each well of a 24 hour well plate at 200,000 cells per well
- Centrifuge specified amount of cells in media (300 g 5 min 4 deg C)
- Decant and Wash with PBS
- Centrifuge and resuspend in media without serum (300 g 5 min 4 deg C)
- 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)
- Add 500 microliters of 20 um solution to 500 microliters media (5 µM label)
- Add 400 microliters of 5 µM solution to 600 microliters of media in eppendorf tube (2 µM label)
- Add 500 microliters of 2 µM to 500 microliters of media in eppendorf tube (1 µM label)
- Add 500 microliters of 1 µM to 500 microliters of media in eppendorf tube (.5 µM label)
- Add 200 microliters of .5 µM to 800 microliters of media in eppendorf tube (.1 µM label)
- Add Daunorubicin solution to cell plate wells
- Add 200 microliters of each concentration to specified cell plate wells
- Put into incubator (label with name/date/what is in each well)
- Incubate for 12 hours
- 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)
- Centrifuge (3 g 5 min 4 deg C)
- Decant media, Resuspend cells in PBS (400 microliter)
- Centrifuge (3 g 5 min 4 deg C)
- Decant, Resuspend cells in HL media with serum (400 microliter)
- Add the samples to a new labeled 24 well plate
- Put back in incubator for 24, 48 hours
- Add 100 microliters poly L-lysine to imaging plate (8 well imaging plate)
- Aspirate/take out cells from culture plate and add to eppendorf tube
- Centrifuge (3 g 5 min 4 deg C)
- Decant Media. Resuspend in 200 microliters PBS
- Centrifuge (3 g 5 min 4 deg C)
- Decant PBS. Resuspend in 200 microliters RPMI 1640 clear media
- Take off poly L-lysine from imaging plate
- Wash imaging plate wells with 200 microliter PBS
- Add 2 microliters 7 A.A.D. to cells in clear media.
- Mix and add to imaging plate
The number obtained multiplied by 10^4 is the cell concentration/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
Sample Plate Layout
| Cell Plate Layout | |||
| 10 uM Dauno | 5 uM Dauno | 2 uM Dauno | Cells only |
| 1 uM Dauno | .5 uM Dauno | .1 uM Dauno | Cells only |
After Incubation (Day One)
Day of Imaging
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