S2 Cells prepS2 Cells prep kdorfman Fri, 07/29/2022 - 20:30
Working with Drosophila S2 Cells
Set incubator for 24C
Notes on S2 cells:
- Do not place in a CO2 incubator.
- S2 cells are happy at room temp, and most happy at ~24C.
- S2 cells get clumpy - and that's OK.
- When ready to split cells, there’s no need to warm the media.
- S2 cells are semi-adherent.
- They adhere to surfaces in a matter of minutes
- They do not need trypsin to dislodge cells from surfaces.
- GFP-Tubulin S2 cells
- Schneider's Drosophila Medium (500ml, 1X) (Gibco cat # 21720-024)
- Heat inactivated FBS
- 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
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)
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)