Culturing S-cells

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 FCS.

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 uL DMSO

Make 1mL aliquots.

Put in -80 in styrofoam for 1 day.

Transfer to liquid nitrogen.