Renew (change medium) cells 24 hrs just before freezing. Cells ought to be healthy and close to confluence sometimes of freezing. They ought to also provide many mitoses apparent by phase microscopy (
day 8 of propagation).
Trypsinize cells as described within the subculture methods. Count cells and see the quantity per vial to become frozen (a great cell phone number per vial is between 1 and 5 x 10 6 ).
After cells happen to be harvested, as described within the subculture methods and counted, cells should then be centrifuged again, the supernatant aspirated and also the cells resuspend within the predetermined amount of medium with 15% FBS (the level of the medium where the cells are resuspended is decided the following: .45 ml medium x vials to become frozen). This ought to be performed inside a stock Lightly pipette cell suspension up and lower a couple of occasions.
Gradually (dropwise) add predetermined amount of DMSO towards the stock tube (50 m l DMSO x vials to become frozen = 10% v/v of DMSO), while lightly mixing the suspension.
Place .5 ml aliquots into cryovials labeled with cell type, passage number, date, quantity of cells and initials.
Transfer vials right into a cryo 1° freezing container and put right into a -80° C freezer overnight. Freeze rate lower to roughly 1° /min.
Transfer vial to liquid nitrogen and record location and all sorts of additional information on vial.
Renew (change medium) cells 24 hrs just before freezing. Cells ought to be healthy and close to confluence sometimes of freezing. They ought to also provide many mitoses apparent by phase microscopy.
Trypsinize cells as described within the subculture methods. Count cells and see the quantity per vial to become frozen (a great cell phone number per vial is between 1 and 5 x 10 6 ).
Resuspend cells within the appropriate amount of Freezing Medium (90% Medium with 15% FBS 10% DMSO). Please work rapidly once cells are resuspended in Freezing Medium, DMSO is toxic to cells.
Place .5 ° 1 ml/cryo vial (10 6 cells) labeled with cell type, passage number, date, quantity of cells and initials.
Transfer vials right into a Cryo 1° freezing container(cryogenic controlled rate freezing container) and immediately put into a -80° C freezer overnight (freeze rate =
1° /min.).
Transfer vial to liquid nitrogen and record location and all sorts of additional information on vial.
