 |
||
|
|
||
| |||||||||||||||
|
|
 ERDC TN-DOER-C30
February 2003
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
"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.
CAUTION: Always wear a protective full-face mask, cryogenic gloves, and lab coat
during the retrieval or storage of cryovials from liquid nitrogen Dewar.
7) Record the number of cryovials, name of cell line, date, technician's initials, and
rack and holder number into the Dewar record book.
Protocol II. Performance of the Assay
I.
Preliminary
Four days are required for performance of the assay. On Day 1, preferably a Monday, cells are
plated into two 96-well microtiter plates and incubated for a day. On Day 2, the cells are dosed
with Ponasterone A standards and environmental extracts. The cells are again incubated for another
48 hours to allow for enzyme ( -galactosidase) induction. On Day 4, the cells are lysed and the
-galactosidase induction is quantitated using a spectrophotometer.
Two 96-well microtiter plates can be set up to test up to a total of 14 environmental samples with
3 replicates each. One-quarter of a 96-well plate is allocated for a complete Ponasterone A
dose-response, while the rest of the wells are assigned to test the environmental samples in
combination with Ponasterone A (4 concentrations). The second 96-well plate will not be necessary
if testing 6 samples or less.
14
|
|
Privacy Statement - Press Release - Copyright Information. - Contact Us - Support Integrated Publishing |
