S2 Cells prep

S2 Cells prep kdorfman Fri, 07/29/2022 - 20:30

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):

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:

Filter sterilize

Prepare Aliquots of S2 Medium for students

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

for instructor flasks, obtain 25 cm2 flasks, label them

pipet to break apart clumps

for student flasks, obtain 12.5 cm2 flasks, label them

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:

(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)