Seeding C2C12 Cells

Seeding C2C12 Cells

Summary

Check how confluent cell dishes are. Mark the one that will be used for seeding (if there is more than one dish) choose the medium confluent dish and trash the others if they are too confluent. As mentioned cells are seeded 10⁵cells per well.

Use same steps as passage

1. Remove old media with vacuum
2. Wash with PBS
3. 1 ml of trypsin into the incubator for two minutes
4. Make sure cell are detached

Create a passage

1. Label the amount of dishes you need (cell type, passage #, date, initials)
2. Add 10 ml of proliferation media for each labeled dish
3. flush trypsinized cells, take up 1:10 ratio (100 ul into 10 ml of media) and spread the five drops in a new plate of cells
4. Shake and place in incubator.

Create a stock of cells+media

• Sterile 15 ml tube (labeled)
• 2 ml of trypsin (1 ml from one plate and another from a second plate)
• 3 ml of prolif. media

(For any volume take one off the number of media for the amount of trypsin added (ie 3 ml of trypsin = 4 ml of media))

Steps for quantifying cells

1. Prepare well plates for seeding, label with time points (no before time point)
2. Check to see all cells are detached
3. Flush with pipette and put 3 ml of media and flush
4. Repeat with each (two round plates total, take 1 ml from each)
5. We know we want 10^5 cells per well times 18 total wells (18 x 10^5) so we use the cell counter to find the amount.
6. *Resuspend cells before using the cell counter*
7. Put 300 ul of cells in the cap (about three drops)
8. Turn on machine (button on back), and place in sensor found in the tissue culture box. If sensor isn’t working try another sensor
9. Hold down plunger in liquid, slowly release. If it is not working place more cells and a new sensor. Do this until screen says: count complete, and use the button to get the results

In this example screen says 6.3 x 10⁵ ml. So you would divide this amount by 18 x 10⁵ (or 18/6.3 = 2.85 ml) so you need 2.85ml of cells to seed 18 wells and 36ml of media total for 18 wells (18x2= 36)

• In a 50 ml tube mix the cells and media

33.2 ml of media (36 ml total- 2.85 total amount of trypsin cells+ media ) 2.85 of cells

1. Load each cell well with 2 ml of mix from the 50 ml tube
2. Place in incubator and swirl