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 ERDC TN-DOER-C10
March 2000
3) Count cell numbers in five sections on each side of the hemacytometer.
4) Total the number of live and the number of dead cells for all 10 sections.
5) Multiply the total number of live cells by a factor of 104 to obtain the number of
live cells/mL in the cell suspension. Likewise, multiply the number of dead cells
by 104 to obtain the number of dead cells/mL. The viability of the cell
suspension is the total number of live cells divided by the total (live and dead)
number of cells.
6) IMPORTANT: A culture with a viability of <95 percent should not be used for
an assay.
D.
FREEZING CELLS
1) Trypsinize, count, and centrifuge cells to form a pellet as described previously.
2) Siphon off supernatant medium.
3) Resuspend cell pellet in an appropriate volume of freezing medium (see Section
II.B, "REAGENT PREPARATION"). The ideal cell concentration is 3 to 5
106 cells/mL.
4) Transfer 1.0 mL cell suspension into prelabeled sterile cryovials. The label should
include the name of cell line, date, cell number, viability, and technician's initials.
5) Place cryogenic vials into the isopropanol freezing chamber and place the freezing
chamber into the -80 C freezer to cool at a rate of 1 C/min overnight.
6) Transfer cryovials from the freezing chamber into a labeled cryobox. Place
cryobox into the liquid nitrogen storage Dewar.
7) CAUTION: Always wear a protective full-face mask, cryogenic gloves, and lab
coat during the retrieval or storage of cryovials from liquid nitrogen Dewar.
8) Record the number of cryovials, name of cell line, date, technician's initials, and
rack and holder number into the Dewar record book.
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