Difference between revisions of "20.109(F19):Incubate with ligand and apply heat treatment for protein denaturation (Day2)"

Introduction

CETSA

Protocols

Part 1: Incubate cells with ligand and harvest for CETSA

Collecting cells

1. Obtain one ~48 h culture of MEF cells in a T25 flask from the 37 °C incubator.
2. 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.
3. After you look at your cells, take the flask to your tissue culture hood to begin the seeding procedure.
4. Aspirate the media from the cells using a sterile Pasteur pipet.
5. 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.
6. With a 2 mL pipet, add 0.5 mL of trypsin to the flask.
7. 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!
8. 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.
9. 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.
10. Transfer the suspended cells into a labeled 15 mL conical tube.
11. 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

Navigation links

Next day: Begin Western blot analysis