This task should take you about an hour. Plan for an hour and a half.

You need to incubate your cells for at least three days before feeding them again. However, observing your cells on a daily basis will add to your growing store of biotechnology knowledge and experience.

Subculture of Substrate Adherent Cells

Materials:

- Confluent cells
- α-MEM medium (serum-free media)
- α-MEM medium + 10% FBS +Antibiotics (complete media)
- 0.25 % trypsin
- T-25 flask
- Hemocytometer

Procedure:

1. Warm fresh culture media to 37 ºC.
2. Aspirate culture medium of cells.
3. Wash cells gently by adding 10 ml of α-MEM medium (serum-free media).
4. Remove serum-free culture medium from cells.
5. Add 1 ml of 0.25% trypsin.
6. Incubate cells for another ~2-5 minutes in trypsin, gently tilting flask and watching for the progress of cell detachment.
7. Gently tap the flask against the surface of hood until cells have completely detached.
8. Immediately add 5 ml of α-MEM medium + 10% FBS +Antibiotics (complete media) to cells.
9. Re-suspend cells well (about 10 x using 10 ml pipette) and take a small aliquot (30ul).
10. Combine aliquot with 30 ul of trypan blue, apply to the hemocytometer and count cells.
11. Determine cell number /ml of your cell suspension.
12. Seed 100,000 cells per new T25-flask.
13. Add 10 ml. complete media to new T25-flask.

Remember: The next step is to come into the lab to practice with the materials, view a demonstration, or to even just ask questions. Also, if you are ready to take the lab final, please inform your instructor. The lab final will be performed by yourself and will be graded with a rubric.

Submission

- Image of cells in a flask, before subculturing
- Image of subcultured cells
- Image of hemocytometer
- Image of calculations and conclusions from lab notebook