StemPro Adipogenesis Differentiation Kit

Description
The StemPro Adipogenesis Differentiation Kit has been developed for the adipogenic differentiation of mesenchymal stem cells (MSCs) in tissue culture
vessels. The kit contains all reagents required for inducing MSCs to be committed to the adipogenesis pathway and generate adipocytes. Using StemPro
Adipogenesis Differentiation Kit in combination with StemPro MSC SFM or MesenPRO RS Medium provides a standardized culture workflow solution
for MSC isolation, expansion, and differentiation into matrix-forming adipocytes.
Product
StemPro Adipogenesis Differentiation Kit
Contains:
StemPro Adipogenesis Differentiation Basal Medium
StemPro Adipogenesis Supplement

Catalog no.

Amount

Storage

Shelf life*

A10070-01

1 kit

A10410-01
A10065-01

100 mL
10 mL

2C to 8C; Protect from light

20C to 5C; In the dark

12 months
12 months

* Shelf life duration is determined from Date of Manufacture.

Product use
For Research Use Only. Not for use in diagnostic procedures.

Important information
StemPro Adipogenesis Supplement is supplied frozen. Thaw prior
to use, as described in Prepare media.
It is normal to see a precipitate formed in the supplement after
thawing. The precipitate does not impact performance of the product.
See Prepare media for guidelines for dissolving the precipitate.
Thawed StemPro Adipogenesis Supplement is stable up to at least
one month at 2C to 8C. Do not refreeze the supplement after
thawing.
Complete StemPro Adipogenesis Differentiation Medium is stable
up to at least one month at 2C to 8C.

Culture vessels: 12-well tissue-culture plates, 16-well CultureWell

slides, 96-well tissue-culture plates, 75 cm2 tissue culture flasks
Temperature range: 36C to 38C.
Incubator atmosphere: Humidified atmosphere of 46% CO2 in air.
Ensure proper gas exchange and minimize exposure of cultures to light.

Important guidelines for adipogenesis differentiation

Expansion culture: Expand primary MSC isolates with StemPro

Safety information
Read the Safety Data Sheets (SDSs) and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and gloves.

Prepare media
Complete Adipogenesis Differentiation Medium
Thaw the supplement in a 372C water bath, and prepare 100 mL of
media according to the following table. Store complete medium at 2C
to 8C in the dark.
It is normal to see a precipitate formed in the supplement after thawing.
To promote dissolution of the precipitate, warm the supplement with
swirling for no more than 30 minutes prior to preparing complete
media. Suspend any remaining precipitate in solution before adding it to
StemPro Adipocyte Differentiation Basal Medium; remaining
precipitate will dissolve completely when mixed with the Basal
Medium and warmed.
Adipogenesis Differentiation Medium

StemPro Adipocyte Differentiation Basal

Medium

StemPro Adipocyte Supplement

Gentamicin reagent (10 mg/mL)

Concentration
1X

Volume
90 mL

1X
5 g/mL

10 mL
50 L

MSC SFM or MesenPRO RS Medium in T-75 or T-150 flasks. We

have successfully used standard growth media of DMEM+10% MSC
Qualified FBS. We recommend refeeding the cultures every 23 days
and passaging every 57 days.
Passaging: We strongly recommend using low-passage MSCs (<8
to 10 passages). Continuously passaged MSCs will gradually lose
their multipotency with increased passage number (>10 passages).
Harvesting: We recommend using TrypLE Express for
enzymatically treating and harvesting MSCs. TrypLE Express is a
recombinant protease that has been demonstrated to be gentle on
MSCs. Overexposure to trypsin will lead to reduced MSC viability
and expansion.
Timing of passaging: Do not let passaged MSCs become
completely confluent, as it can reduce multipotency of MSCs.
Passage cultures when they reach 6080% confluency, cell viability
is at least 90%, and the growth rate is in mid-logarithmic phase.
Seeding density: For expansion, we recommend a seeding density
of 3 103 to 5 103 viable cells/cm2 with MesenPRO RS Medium
or 1 104 viable cells/cm2 with StemPro MSC SFM.
Confluency: Expanding MSCs in growth medium for 24 days
before refeeding with Complete Adipogenesis Differentiation
Medium can enhance adipogenesis.

Adipogenesis differentiation
MSC growth medium

1.

Prepare MSC growth medium according to the following table.

MSC growth medium (500 mL)
DMEM low glucose
MSC-qualified FBS

GlutaMAX -I (200 mM)

Gentamicin reagent (10 mg/mL)

Final concentration

10%
2 mM
5 g/mL

Volume
445 mL
50 mL
5 mL
250 L

2.
3.

Culture conditions
Media: StemPro Adipogenesis Differentiation Medium.
Cell line: Human mesenchymal stem cells.
Culture type: Adherent
Publication Number MAN0000693

4.
5.

Observe cell monolayer from basal cultures expanded in StemPro

MSC SFM, MesenPRO RS medium, or standard growth medium
(DMEM+10% FBS) to ensure mid-log growth phase confluence
(6080%). Aspirate medium and floating cells from culture flask
and discard.
Add 510 mL DPBS. Gently rinse cell monolayer.
Remove DPBS, add 57 mL of pre-warmed TrypLE Express to
flask and completely coat culture surface. Incubate for
58 minutes at 36C to 38C or until cells have fully detached.
Gently pipet detached cells into a single cell solution and verify on
inverted microscope.
Remove cell suspension from flask, transfer into a centrifuge tube,
and pellet cells at 100 g for 510 minutes.
Rev. 2.00

6.

Dettermine cell viab

bility and total ceell density using
g Trypan Blue
Stain and an electro
onic (Coulter Co
ounter) or manuaal
mocytometer) ceell counting metthod.
(hem
7. Ressuspend the pelleet in an appropriiate volume of prre-warmed
MS
SC Growth Mediium (see Preparre media, page 1).
1
8. Seeed MSCs into cu
ulture vessels at 1 104 cells/cm2. For classical
staiin differentiation
n assay, seed into
o a 12-well platee. For gene
exppression profile studies,
s
seed into
o a T-75 flask. For
F
imm
munocytochemisstry studies, seed
d into a 16-well CultureWell
chaambered coverglass or 96-well plate.
9. Incuubate the cells in
n MSC Growth Medium
M
at 36C
C to 38C in a
hum
midified atmosph
here of 46% CO
O2 for a minimum
m of 2 hours up
to 4 days.
10. Repplace media with
h pre-warmed Co
omplete Adipog
genesis
Diffferentiation Med
dium and continue incubation. MSCs
M
will
conntinue to undergo
o limited expanssion as they diffeerentiate under
adippogenic conditio
ons. Refeed cultu
ures every
344 days.
11. Aftter specific perio
ods of cultivation
n, adipogenic culltures can be
processed for Oil Red
R O or LipidTO
OX staining (beginning at 7
f method), gen
ne expression
14 ddays; see the folllowing section for
anaalysis, or protein detection.

HCS LipidTOX Gre

een neutral lip
pid stain anallysis
1.

2.

3.

Aftter 7 days or long

ger under differeentiating conditions, remove the
meddia from the 16-well CultureWell or 96-well tiissue culture
platte and rinse oncee with DPBS. Fiix cells with 4% formaldehyde
soluution for 30 min
nutes.
Aftter fixation, rinse wells twice with
h DPBS, apply 1:100 dilution
LippidTOX Green and incubate for
1530 minutes. You
u can add 1:4000
0 Hoechst 33342 as a nuclear
couunterstain.
Rinnse twice with DPBS, apply Slow
wFade Gold to the wells,
visuualize under fluo
orescent microsccope and capturee images for
quaalitative or quanttitative analysis.

Figure
e 2 Analysis of M
MSCs cultured in S
StemPro Adipog
genesis
Differe
entiation Medium
m demonstrated d
differentiation into
o adipogenic
lineag
ges by FABP4 an
nd CD36 immuno
ostaining.

Rela
ated products
Product

CTS StemPRO MS
SC SFM

Stem
mPRO Human A
Adipose-Derived S
Stem Cell Kit

Stem
mPRO Osteogen
nesis Differentiattion Kit

Mese
enPRO RS Me dium
FBS,, MSC-Qualified (non-US)

Gluta
aMAX -I
Genttamicin reagent ((10 mg/mL)

Tryp LE Express
++
++
DPB S without Ca a
and Mg

HCS
S LipidTOX Gree
en neutral lipid

Slow
wFade Gold antiffade reagent

Cultu
ureWell chambe
ered coverglass
Trypa
an Blue Solution,, 0.4%

Catalog no.
A10332
R7788
A10072-0
01
12746
12662
35050
15710
12604
14190
H34475
S2828
C37005
15250

Expl anation of syymbols and wa

arnings
The syymbols present onn the product label are explained beloow:

Imagess of cells in SttemPro Adip

pogenesis Diffferentiation
Medium
m

Figure 1 Analysis of MSC

Cs cultured in Stem
mPro Adipogene
esis
emonstrated differentiation into adipogenic lineage
Differentiation Medium de

ed O and HCS LipidTOX Green neutral lipid stain

ning.
by Oil Re

Tem
mperature Limitation

Batch code

Use By:

Catalog number

C
Caution, consult
accom
mpanying documents

Consult instructions
for use

Keep away
from light

Sterilized using aseptic

processing technique
es

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warranty
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ortant licensin
ng information
n
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ditional technica
al information such
s
as Safety Data Sheets (SDS), Certifica
ates of Analysiss, visit www.life
etechnologies.ccom/support
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her assistance, email techsu
upport@lifetec
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