Culturing S-cells

Culturing S-cells kdorfman Fri, 05/29/2020 - 15:44

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.