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
- cells are neomycin resistant
- Induce 1 day prior to imaging
- to image put on previously prepared ConA coated coverslips. Image 30 min after plating on Con-A coated dishes or Con-A coated coverslips
To make medium for growth of Drosphila cells:
Schneider’s medium + 10% heat-inactivated FBS.
Freezing S2 cells
Spin down a 3-4 day old 75 cm2 confluent flask. 1200 RPM, 5 min
Remove conditioned medium
Resuspend one flask’s worth of cell pellet in “freezing medium”
- 1.8 mL fresh medium (including antibiotic and serum)
- 1.8 conditioned
- 400 µL DMSO
Make 1mL aliquots.
Put in -80 in styrofoam for 1 day. (or use Mr. Frosty)
Transfer to liquid nitrogen.