Difference between revisions of "20.109(F19):Incubate with ligand and apply heat treatment for protein denaturation (Day2)"
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==Protocols== | ==Protocols== | ||
− | ===Part 1: Incubate cells with ligand=== | + | ===Part 1: Incubate cells with ligand and harvest for CETSA=== |
'''Collecting cells''' | '''Collecting cells''' |
Revision as of 20:55, 1 September 2019
Contents
Introduction
CETSA
Protocols
Part 1: Incubate cells with ligand and harvest for CETSA
Collecting cells
- Obtain one ~48 h culture of MEF cells in a T25 flask from the 37 °C incubator.
- Examine your cell cultures after you remove the flask from the incubator.
- Look first at the color and clarity of the media. Fresh media is reddish-orange in color and if the media in your flask is yellow or cloudy, it could mean that the cells are overgrown, contaminated, or starved for CO2.
- Next, look at the cells using the inverted microscope. Note their shape, arrangement, and how densely the cells cover the surface of the flask.
- After you look at your cells, take the flask to your tissue culture hood to begin the seeding procedure.
- Aspirate the media from the cells using a sterile Pasteur pipet.
- Wash the cells by adding 2 mL PBS using a 5 mL pipet. Slightly tip the flask back and forth to rinse the cells then aspirate the PBS with a Pasteur pipet.
- With a 2 mL pipet, add 0.5 mL of trypsin to the flask.
- Tip the flask in each direction to distribute the trypsin evenly then incubate the cells at 37°C for 2 minutes using a timer.
- This is a great time to clear out your trash and read ahead!
- Retrieve your flask from the incubator and firmly tap the bottom to dislodge the cells.
- Check your cells using the microscope to ensure they are dislodged. They should appear round and move freely.
- If your cells are not detached from the flask, incubate at 37 °C for an additional minute.
- When your cells are dislodged, move your flask back into the tissue culture hood and add 3 mL of media to the cells then pipet the liquid up and down (“triturate”) to break up cells that are clumped together and suspend them in the liquid.
- Note: do not take up or release all the liquid, in order to avoid bubbles.
- Transfer the suspended cells into a labeled 15 mL conical tube.
- Transfer 90 μL of your cell suspension from the 15 mL conical tube into a labeled eppendorf tube.
- This aliquot will be used to determine the concentration of the cell culture.
- Be sure to cap your conical tube and eppendorf tube after you transfer your cells.
Part 2: Apply heat treatment and snap freeze cells
Reagents list
Next day: Begin Western blot analysis