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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
16634 (Sigma)
(IBMX)
17018 (Sigma) (DMEM high glucose, 10% FBS,
1% Penicillin)
Method:
2. Vortex
Note: Dexamethasone is light sensitive; store it in brown tube or dark place, at 4ºC
6. Vortex well.
Note: IBMX is light sensitive; store it in brown tube or dark place, at 4ºC.
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.
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:
Concentration Concentration
Note:
1. Final volume of differentiation media will be 100 ml; so, take out 1200 µl of complete media and then add Dexamethasone,
• 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
• Keep the cells for another 48 hours in this state, to arrest the cell division.
6 well plate 5 4 ml
0.8 x10 cells/well
24 well plate 5 1 ml
0.2 x10 cells/well
(72 hours)
• 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
• After Day 8, lipid droplets inside the cells will be gradually visible and around day 14, lipid droplets will become larger. After
• Cells can be kept for around 1 month in this condition by changing media in every 2 days.
Note:
1. During the differentiation procedure, many times media are needed to be changed. So, precaution should be taken to prevent contamination.
Adipocyte differentiation can be confirmed by staining the lipid droplets with Oil Red O.
Materials
Method
2. Stir overnight
4. store at 4°C
1. Mix 6 parts of Oil Red O stock solution in 4 parts DW mix and let sit at room temperature for 20 min.
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
5. Wrap parafilm around the plate to prevent from drying and cover with aluminum foil.
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 .
O.D Measurement:
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.
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).
isopropanol isopropanol
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Partho
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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