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Life Science: 3T3-L1 Adipocyte Differentiation Protocol

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Sunday, July 19, 2009

3T3-L1 Adipocyte Differentiation Protocol

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Life Science: 3T3-L1 Adipocyte Differentiation Protocol

3T3-L1 Adipocyte Differentiation Protocol

Obesity is an increasing problem worldwide and induces many diseases like diabetes, atherosclerosis and other metabolic syndrome. So the

knowledge about the process of adipogenesis and formation of adipose tissue is very important. For the better understanding of these

processes, an in vitro model is necessary. Few cell lines can undergo Adipocyte differentiation; of them mouse 3T3-L1 cell is well

characterized cell line for adipogenic assay. In normal condition 3T3-L1 preadipocyte cells have fibroblastic phenotype. When this cell is

treated with differentiation media, they accumulate lipid droplet inside the cell and achieve Adipocyte phenotype.

Adipocyte Differentiation takes around two weeks and differentiation can be confirmed by Oil Red O Staining.

Materials

Name Catalogue No. (brand) Name Catalogue No. (brand)

Dexamethasone D2915 (Sigma) Insulin 090-03446 (Wako)

16634 (Sigma)

3-Isobutyl-1-methylxanthine 095-03413 (Wako)/ Complete media

(IBMX)
17018 (Sigma) (DMEM high glucose, 10% FBS,

1% Penicillin)

Method:

Adipocyte differentiation media Preparation:

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Life Science: 3T3-L1 Adipocyte Differentiation Protocol

(1 µM Dexamethasone, 0.5mM IBMX, 10 µg/ml Insulin, Complete media).

• Dexamethasone Preparation (Stock solution):

1. Dissolve 0.39 mg Dexamethasone in 1 ml DW

2. Vortex

3. Filter through 0.22 µm syringe filter

Final Concentration will be 0.994 mM.

Note: Dexamethasone is light sensitive; store it in brown tube or dark place, at 4ºC

• IBMX Preparation (Stock Solution):

1. Take 11.5 mg in 2ml vial.

2. Add 500µl DW.

3. Add 1N NaOH (around 100 to 200 µl)

4. Vortex it, dissolve well.

5. Add DW to make the final volume 1 ml.

6. Vortex well.

7. Filter through 0.22µm syringe filter.

Final concentration will be 51.8 mM.

Note: IBMX is light sensitive; store it in brown tube or dark place, at 4ºC.

• Insulin Preparation (Stock Solution):

1. Take 10 mg insulin powder in 2 ml Vial.


2. Add 500µl DW

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Life Science: 3T3-L1 Adipocyte Differentiation Protocol

3. Vortex
4. Add 0.02mM HCl (around 100 to 200 µl) to dissolve insulin.
5. Vortex well.
6. Add DW to make final volume 1 ml.
7. Filter through 0.22µm syringe filter.

Final Concentration will be 10 mg/ml

Note: Insulin is light sensitive; store it in brown tube or dark place, at 4ºC.

Add the following reagents to 100 ml of complete media on the day of differentiation and mix well:

Reagent Stock Volume Added Final

Concentration Concentration

Dexamethasone 0.994 mM 100 µl 1 µM

IBMX 51.8 mM 1 ml 0.5 mM

Insulin 10 mg/ml 100 µl 10 µg/ml

Note:

1. Final volume of differentiation media will be 100 ml; so, take out 1200 µl of complete media and then add Dexamethasone,

IBMX and insulin. Mix well by pipetting.

2. Always use freshly prepared media.

Seeding of 3T3-L1 preadipocyte cell line for induction of differentiation

• When the 3T3 preadipocyte cells are around 70% to 80% confluent, harvest the cells from tissue culture flask by

Trypsinization.

• Seed the cells in complete media on desired tissue culture vessel like 96/24/12/6 well plate. (See below for cell seeding

density, volume of media and differentiation schedule).

• Let the cells grow to 100% Confluency (2 days).

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Life Science: 3T3-L1 Adipocyte Differentiation Protocol

• Keep the cells for another 48 hours in this state, to arrest the cell division.

• Then treat with Adipocyte differentiation media.

Culture vessel Cell Density Volume of media

6 well plate 5 4 ml
0.8 x10 cells/well

24 well plate 5 1 ml
0.2 x10 cells/well

96 well plate 3 100 µl


2 x10 cells/well

3T3-L1 adipocyte differentiation schedule:

Preadipocyte culture Post Confluency Adipocyte Differentiation Adipocyte Maturation

Day -1 Day -2 48 hours Day 0 to Day 3 Day 4

(72 hours)

Feed Complete Media Feed Adipocyte Feed Adipocyte Maturation

Differentiation Media Media in every 2 days

• After post confluency, feed the cells with adipocyte differentiation media (1 µM Dexamethasone, 0.5 mM 3-Isobutyl-1-

methylxanthine, 10 µg/ml Insulin and complete media) at day 0. Keep the cells for 72 hours in this condition.

• After 72 hours, discard all the media by gentle pipetting. In this stage, cells can be easily detached from plate. (So, pipette

gently).

• Add Adipocyte maturation media (Complete media and 10 µg/ml Insulin) at day 4. Change the media in every 2 days. During

changing the media don’t discard all the media (for example, if you have 2 ml media. Discard 1 ml from it and add 1 ml fresh

Adipocyte maturation media).

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Life Science: 3T3-L1 Adipocyte Differentiation Protocol

• After Day 8, lipid droplets inside the cells will be gradually visible and around day 14, lipid droplets will become larger. After

day 8, cells are ready for assay.

• Cells can be kept for around 1 month in this condition by changing media in every 2 days.

• Differentiation can be confirmed by Oil Red O staining.

Note:

1. During the differentiation procedure, many times media are needed to be changed. So, precaution should be taken to prevent contamination.

Oil Red O Staining of Adipocyte differentiated cells

Adipocyte differentiation can be confirmed by staining the lipid droplets with Oil Red O.

Materials

Name Catalogue No. (Brand) Name Catalogue. (Brand)

Oil Red O O-0625 (sigma) 10% Formalin in PBS

Isopropanol 100% Isopropanol 60%

Method

Oil Red O stock solution preparation:

1. Dissolve 0.7 g Oil Red O powder in 200 ml 100% Isopropanol.

2. Stir overnight

3. Filter through 0.22 μm membrane filter

4. store at 4°C

Oil Red O Working solution Preparation:

1. Mix 6 parts of Oil Red O stock solution in 4 parts DW mix and let sit at room temperature for 20 min.

2. Filter through 0.22 μm membrane filter.

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Life Science: 3T3-L1 Adipocyte Differentiation Protocol

Note: Oil Red O working solution is stable around 1 hour.

Oil Red O staining:

1. Remove most of the medium

2. Wash 1x with PBS.

3. Add 10% formalin and incubate 5 min at room temperature.

4. Discard formalin and add the same volume of fresh formalin. Incubate at least 1 hour, or longer (Cells can be kept in formalin for a

couple of days before staining.).

5. Wrap parafilm around the plate to prevent from drying and cover with aluminum foil.

6. Remove all the formalin.

7. Wash wells with 60% isopropanol.

8. Let the wells dry completely.

9. Add Oil Red O working solution and incubate for 10 min at room temperature (do not touch the walls of the wells)

10. Remove all Oil Red O and immediately add DW, wash 4 times with DW .

11. Take pictures if desired.

O.D Measurement:

1. Remove all water and let dry

2. Elute Oil Red O by adding 100% isopropanol, incubate about 10 min at room temperature (can be longer)

3. Pipette the isopropanol with Oil Red O up and down several times to be sure that all Oil Red O is in the solution.

4. Transfer 1 ml solution to cuvette

5. Measure OD at 520 nm in spectrophotometer,

6. As blank use 100% isopropanol.

7. As control use isopropanol from empty well stained. First, add Oil Red O working solution in 6 well plate (one or two well is enough).

Incubate 10 min at room temperature. Discard Oil Red O. Add 100% isopropanol and pipette the isopropanol with Oil Red O up and

down several times to be sure that all Oil Red O is in the solution.

8. Result: O.D of Non Differentiated cells: 0.05 and OD of Well Differentiated Cells: 0.7 to 1.0 (in 6 well plate).

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Life Science: 3T3-L1 Adipocyte Differentiation Protocol

Plate Formalin 60% Oil Red O 100%

isopropanol isopropanol

24 well plate 500 µl 500 µl 200 µl 750 µl

12 well plate 1 ml 1 ml 400 µl 1.5 ml

6 well plate 2.4 ml 2.4 ml 1 ml 3.6 ml

posted by Partho at 4:50 PM

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Partho

Seoul, Seoul, South Korea

Hello.... I am Partho, from Bangladesh. Currently I am living in South Korea. I have completed my graduation in

Biotechnology and Genetic Engineering from Bangladesh. Now I am a graduate student in Hanyang University, Korea. Here,

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