S2 Cells prep

Working with Drosophila S2 Cells

Set incubator for 24C

Notes on S2 cells:

  1. Do not place in a CO2 incubator.
  2. S2 cells are happy at room temp, and most happy at ~24C.
  3. S2 cells get clumpy - and that's OK.
  4. When ready to split cells, there’s no need to warm the media.
  5. S2 cells are semi-adherent.
  6. They adhere to surfaces in a matter of minutes
  7. They do not need trypsin to dislodge cells from surfaces.

Materials (sterile):

  • GFP-Tubulin S2 cells
  • Schneider's Drosophila Medium (500ml, 1X) (Gibco cat # 21720-024)
  • Heat inactivated FBS
  • anti-microbial/anti-mycotic
  • Culture Flasks (25 cm2, non-vented cap)(Fisher Cat # FB012935)
  • (Culture Flasks for students will be 12.5 cm2)(Fisher Cat # FB012933)
  • 35 mm glass bottom dishes with 14mm bottom well (www.cellvis.com Cat # D35-14-1.5-N)
  • Con-A treated dishes and Non-Con-A treated will be used in class

Preparing medium:

from fresh 500 ml bottle of S2 medium, remove 55 ml (place 50 ml into a sterile conical and label "serum-free" for later in the semester)

Add to remaining 445 ml S2 medium:

  • 50 ml of heat-inactivated FBS
  • 5ml anti/anti (0.5X final)

Filter sterilize

Prepare Aliquots of S2 Medium for students

To Split cells, (~2x/wk):

for instructor flasks, obtain 25 cm2 flasks, label them

  • 5ml fresh media + additives
  • 1 ml cells

pipet to break apart clumps

for student flasks, obtain 12.5 cm2 flasks, label them

  • 2.5 ml fresh medium
  • 0.5 ml of cells (maximum, no more than 3ml total in these flasks)

For viewing Tubulin-GFP in dishes

20 minutes before the first class, plate cells unto Con-A treated 35 mm glass bottom plates (n=5) and non-treated 35 mm glass bottom plates (n=5). For the rest of the semester all glass-bottom dishes need to be ConA-coated

for each plate, add:

  • 450 ul fresh medium with additives
  • 50-100 ul cells

(final total volume will be 500 ul.) After 15- 20 minutes the cells will adhere to the flask and can be viewed.

Students can add a drop of nuc blue (special Hoechst) to view nuclei (Molecular Probes / Life Technologies Cat # R37605)