7717

• UCREFRP Catalog Number: 7717
• Author: Rosenfeld, M. J.
• Title: Genetic Analysis of the Colorado River Gila
• USFW Year: n.d.
• USFW - Doc Type: Non-fatal Sampling, Cell Culture, Chromosomal Analysis and Electrophoresis.
• Copyright Material: NO

eX~lrpa~~d and rInsed well wIth sterIle phosphate-buttered saline

solution (PBS)

containing 50 ug/ml neomycin (Sigma) and 20 IU/ml

nystatin (Sigma).

The tissue was then placed in a stor~ge

solution

L15 Medium

(Sigma) containing 201. iron-supplemented

calf serum (Sterile Systems)~ 50 ug/ml

kanamycin

(Sigma)~ 50

ug/ml neomycin~

100 IU/ml penicillin (Sigma)~ 100 ug/ml strepto-

mycin (Sigma)

and 20 IU/ml nystatin;

sterile filtered in 4 ml

aliquots into 12 X 75 mm capped tubes (Falcon) -- and kept on ~et

ice. Fin clips yielded live cells for at least 7 d when sto~ed

in this manner-.

All cell culture procedures were done under sterile con~i-

tions in a laminar flow hood. Each fin clip was removed from the

storage solution,

washed with agitation in 70'l. ethanol for 15+25

sec (Uwa et al., 1981>, rinsed in 2 changes of PBS~ and planted

in a 25 cm2 culture flask (Falcon).

After keeping the fl.sk

perpendicular for 20-40 min to promote adhesion of tissue to the

vessel surface,

4 ml of culture medium was added -- Dulbecco's

Modified Eagle Medium <Sigma) containing 20'l. iron-supplemenied

calf

I
and 100 ug/ml streptomycin.
I

100

IU/ml

penicillin

serum,

Each flask was gassed with 5'l. CO2. Medium was changed after 4 d

and subsequently every other day. All cultures were incubated at

300 c.

A secondary culture was set-L~ when fibroblast gro~th

e:.: tended about

1 cm from the fin explant~ usually within ~-4

wk.

(1:250 Difco; ~.5

Fibroblasts were suspended with trypsin

mq/fTIJ)

0.1-1 d

dl~:,odlurn

ethylenedjamlnetetracetic acid

(~;lgma;

i
~

, '?
(~). ..;..