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1.. Add sufficient Gelatin Solution into the cu
ulture vesse
el to complletely coverr its base.
2.. Swirl unttil Gelatin Solution
S
coa
ats the entire base of vessel. Lett it sit for att least 30
minutes at
a room tem
mperature..
3.. Aspirate off all of th
he Gelatin S
Solution and allow the
e residual a mount to evaporate
e
ng the vesse
el sitting op
pen in the laminar
l
flow
w hood/bio
ological safe
ety cabinet
by leavin
for no mo
ore than 30
0 minutes.
e it has dried.
4.. Enclose the
t
culture vessel once
sks can be
Note: Gelattinized dis
shes or flas
e stored at 4°C for n
no more th
han 2
w
weeks, prov
vided they
y remain s
sterile.
Th
hawing and Establishing W
Wistar Ratt ADSCs
m the fully supplemen
s
ted (complete) OriCelllTM Rat ADS
SC Growth Medium to
o
1.. Pre-warm
37°C.
2.. Add 9 mL
L of OriCellTM Rat ADS
SC Growth Medium
M
to a 15 mL co
onical tube..
3.. Remove the cryovia
al of OriCelllTM Wistar Rat
R ADSCs from
f
liquid nitrogen.
4.. Quickly thaw
t
the via
al in a 37°C
C water batth until the last ice cry
ystal disapp
pears. For
optimal results,
r
be sure to fini sh the thaw
wing proced
dure within
n 3 minutes
s. Be
careful not to subm
merge the en
ntire vial. Maximum cell
c viability
y is depend
dent on the
d complete thawing off frozen cells.
rapid and
ess than op
ptimal if the
e cells are thawed
t
forr more than
n 3 minutes
s.
Note: Resultts will be le
N
5.. As soon as
a the cells
s are complletely thawed, disinfec
ct the outsiide of the cryovial
c
with 70%
% v/v ethan
nol.
TM
Ratt
6.. Use a pip
pette to transfer the ccells to the 15 mL coniical tube co
ontaining OriCell
O
ADSC Grrowth Mediu
um inside a biosafety cabinet. Be
B careful n
not to introd
duce any
bubbles during
d
the transfer prrocess.
7.. Rinse the
e vial with 1 mL of the
e medium to
t reduce cell loss. Su bsequently
y transfer
this 1 mL
L of cell sus
spension in
nto the conical tube.
8.. Gently mix
m the cell suspension
n by slowly pipetting up
u and dow
wn. Be care
eful not to
introduce
e any bubbles.
9.. Centrifug
ge the cell suspension
s
at 250 x g for 5 minu
utes.
10
0. Carefully
y aspirate off as much of the supernatant as
s possible a
and add 2-3
3 mL of
fresh OriCellTM Rat ADSC
A
Grow
wth Medium
m (pre-warm
med to 37°C
C).
11
1. Gently re
esuspend th
he cells in O
OriCellTM Ra
at ADSC Grrowth Mediu
um.
n and add sufficient
12
2. Seed the
e cells into a T25 flask
k pre-coated
d with Gela
atin Solution
OriCellTM Rat ADSC Growth Me
edium. Gen
ntly rock th
he culture fllask to evenly
distribute
e the cells.
13
3. Incubate
e the flask at
a 37°C ins ide a 5% CO
C 2 humidiffied incubattor.
14
4. The nextt day, chang
ge the med
dium with fresh
f
growth medium (pre-warmed to 37°C
C).
15
5. Change the
t
growth medium ev
very two da
ays until the cells are 80% confluent
thereafte
er.
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