Freezing and Thawing S2 Cells

• SOP- Cryogenic Materials
• SOP- Biosafety Cabinets

1. Use and early passage (1-10) of exponentially growing (about ~10^7 cells).
2. Detach by pipeting a stream of media over the cells
3. Transfer into a 15 mL falcon tube
4. Spin at 500g for 10 min, save the supernatant
5. Prepare freezing media:
   1. Add 4.5 mL supernatant
   2. Add 4.5 mL fresh media
   3. Add 100 uL sterile DMSO
6. Resuspend to 1/10th original culture volume
7. Dispense 0.5 mL into sterile cryovials
8. Place in isopropanol freezing chamber and store at -80 for about a day
9. Transfer to liquid nitrogen
10. Cells should be viable for >5 years

see PMID 18388942