Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
2. ? Place the flasks on ice and allow them to equilibrate for at least 30 minutes.? Gently scrap the bottom of each flask with a cell scrapper to detach adherent cysts.?
3.?? Transfer the cyst suspensions to 15 ml plastic centrifuge tubes and centrifuge at 800 x g for 5 minutes.? Place the concentrated cyst suspension on ice. Adjust the concentration of cells to 2 x 105/ml in fresh cold medium.
4.? Mix the cell preparation and the cryoprotective solution in equal portions. ??Keep the suspension on ice.
5.?? Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. ? Place the vials in a Nalgene 1°C freezing apparatus.? Place the apparatus at -80°C for 2.0 to 2.5 hours and then plunge ampules into liquid nitrogen.? (The cooling rate in this apparatus is approximately -1°C/min.) ?
7. ? Ampules are stored in either the vapor or liquid phase of a
nitrogen refrigerator.
8. ? To establish a culture from the frozen state refer to the
????? instructions above.
