If you only want to change your medium: it’s pretty easy.

Aspirate the supernatant from the surface of the liquid, but not too close to the cell layer. Don’t worry, your cells are adherent so they will stick to the bottom of the flask while you do your thing, as long as you are not too harsh. Once you’re done, replace with new medium (check that you have pre-warmed it in the incubator/water bath).

If you want to subculture (your cell density is too high): you’re going to have a bit more fun.

First, you will need to detach them from your flask/well enzymatically or mechanically to perform a cell count.

• To detach your cells enzymatically:

  you first remove your supernatant and wash with 50mL PBS (an ionic solution, sterile please!). This is important because enzymatic solutions won’t work if there is serum from your medium left. You then add your enzymatic solution (2-3mL) and incubate (room temperature or incubator) until detached, check under the microscope regularly (5-10min should be enough). Do not leave them in contact with the enzymes for too long, or they will start dying! Add twice as much growth medium as enzymatic solution (4-6mL) to stop the reaction and centrifuge your cells at the recommended centrifugation speed. When done, aspirate the supernatant being careful not to disrupt the pellet. Resuspend in a small amount of growth medium (10-20mL should be enough) and take a sample to do the cell count (click here for an explanation).

• To detach your cells mechanically:

  remove the supernatant and add new medium (previously warmed up). Use a cell scraper to gently detach your cells from the bottom surface of the flask. Check that you have detached all of them (by inspecting visually the bottom of the flask or if it’s difficult to tell, under a microscope, but remember to close the flask with the cap before taking it out!) and that the cell suspension is homogeneous before taking a sample to do the cell count (click here for an explanation).

Top up with medium to the final volume desired, place in new flasks (according to recommended seeding density) and back to the incubator.