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OriCellTTM DOG ADSC
A
DI
IFFEREN
NTIATIO
ON USIN
NG OriCe
ellTM
DIFFER
RENTIAT
TION ME
EDIA
OriCellTM Dog
g ADSCs ca
an differenttiate into a variety of cell
c types in
ncluding os
steocytes,
dipocytes, and
a
chondrrocytes.
ad
O
Osteogenic Differen
ntiation
M
Materials Required
R
OriCellTM Mes
senchymal Stem Cell O
Osteogenic
c Differentia
ation Mediu
um (Cat. No
o. GUXMX0021)
90
sis Protoco
ol
Osteogenes
Note: The protocol listed below iss for 6-welll tissue cultture platess.
N
1.. Culture the
t
OriCellTM Dog ADSCs in OriCe
ellTM Mesenc
chymal Ste
em Cell Gro
owth
Medium at
a 37°C in a 5% CO2 humidified incubator.
2.. When cells are apprroximately 80-90% co
onfluent, th
hey can be dissociated
d with
psin-0.04%EDTA (Cat. N
No. TEDTA-10001).
0.25%Tryp
2
in
3.. Reseed the MSCs in
n the growtth medium at 2×104 cells/cm
c
n a 6-well tissue
p
pre-co
oated with 0
0.1% gelatin solution..
culture plate
4.. Incubate
e the cells at
a 37°C in a 5% CO2 humidified
h
incubator.
5.. When cellls are apprroximately 60-70% co
onfluent, ca
arefully asp
pirate off the growth
medium from each well and ad
dd 2 mL of OriCellTM Mesenchyma
M
ell
al Stem Ce
nic Differentiation Med
dium.
Osteogen
6.. Feed cells every 3 days
d
for 2-3
3 weeks by
y completely
y replacing the mediu
um with
enchymal S
Stem Cell Osteogenic
O
Differentiat
D
tion Medium
m (prefresh OriCellTM Mese
warmed to 37°C).
7.. After 2-3
3 weeks of differentiat
d
tion, cells can be fixed
d and staine
ed with aliz
zarin red S.
event osteo
oblasts from
m detaching, it is reco
ommended to change half of the
N
Note: To pre
m
medium every two days
s before an
nalysis.
Allizarin Red
d S Stainin
ng Analysiis
1.
After the
e cells have differentia
ated, remov
ve the osteo
ogenic diffe
erentiation medium
from the wells and rinse with 1x phospha
ate-buffered saline (PB
BS). Fix ce
ells with 2
mL of 4%
% formaldehyde solutiion for 30 minutes.
m
2.
Rinse we
ells twice with 1x PBS.. Stain the cells with 1 mL alizarrin red S working
solution for
f 3-5 min
nutes.
3.
Rinse we
ells 2-3 time
es with 1x PBS.
4.
Cells can
n now be vis
sualized an
nd analyzed
d under a microscope.
m
IMPI0053A2 CA
AXMDͲ01001
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