Cells are in suspension -- they are not dead!
To make conditioned medium:
- spin cells to bottom of conical tube (be gentle!!).
- Save the supernatant in a labeled tube that has the date and is labeled “conditioned medium”.
- Store extra in fridge.
To grow cells:
- Grow cells at room temperature, no CO2.
Passage every 3-4 days. Dilute cells 1:5 each split and use 20% conditioned medium.
Put cells from flask in tube; spin; take off and save supernatant (this is your conditioned medium!)
- Resuspend pellet (gently) in 5 mL medium.
- Add to clean flask
- 1 mL conditioned medium (supernatant)
- 1 mL cell suspension
- 3 mL fresh medium
- To induce gene expression (e.g, tubulin) add copper sulfate to the medium
To make medium for growth of Drosphila cells:
Schneider's medium + 10% heat-inactivated FCS.
Freezing S2 cells
Spin down a 3-4 day old flask.
Resuspend in 4 mL of S2 medium with serum and antibiotic.
Add 1mL DMSO (20% final).
Make 1mL aliquots.
Put in -80 in styrofoam for 1 day.
Transfer to liquid nitrogen.