3T3-L1 Differentiation Protocol
Step 1: 3T3-L1细胞传代于35或60-mm培养板中，使用DMEM-F12培养基培养2 day（以此为基点，即：诱导分化的第0 day）。

Step2: 使用诱导液I诱导3T3-L1分化。

加入配制好的诱导液I于
3T3-L1细胞中，培养2 day（诱导分化的第2 day）。

诱导液I：0.5m M IBMX;
0.25 μM地塞米松；
1μg/ml insulin;
含10% FBS 的DMEM-F12。

Step3: 使用无血清培养基清洗细胞，去除残余的IBMX和地塞米松。

使用诱导液II诱导细胞分化，并培养2 day （诱导分化的第4 day）。

诱导液II：
1μg/ml insulin;
含10% FBS 的DMEM-F12。

Step4: 最后用普通DMEM-F12培养基培养并每2 day 换一次液。

Step5: 每次实验前，用serum-free DMEM-F12 或KRP buffer 培养单层细胞2 h。

Step6: 用于实验的脂肪细胞应该在诱导分化后的8-12 day之间为宜。

这样的条件下≥ 95%的细胞表现为脂肪细胞的表型。

Step7: Oil Red O staining
鉴定分化后的脂肪细胞内的脂质，可以使用油红染色。

细胞用PBS
轻轻冲洗2次，使用10 %的formalin或4% paraformaldehyde 室温固定30 min。

固定后的细胞使用新配制的Oil Red O solution 染色（注意避光）1 h，之后只用蒸馏水轻轻冲洗3次，最后观察结果。

Oil Red O solution 配制：0.5%（W/V）的Oil Red O-异丙醇溶液。

取0.5%的Oil Red O-异丙醇溶液6份，加入4份的蒸馏水，配制成Oil Red O solution。

Step8: 获得结果并进行后期处理。

Other protocol can be used:
美国密歇根大学的3T3-L1细胞分化的PROTOCOL(非常详细)
3T3-L1 Differentiation Protocol
MATERIALS
Dulbecco's Modified Eagles Medium (DMEM; GibcoBRL-Cat# 11965-084: high glucose, with L-glutamine, with pyroxidine HCl, without sodium pyruvate)
Calf Serum (GibcoBRL-Cat#16170-078/Lot #1060198)
Fetal Bovine Serum (GibcoBRL-Cat# 10437-028/Lot # 1026566)-filter sterilize (0.22um filter) before mixed into DMEM
Isobutylmethylxanthine (IBMX; Sigma I-7018)
Dexamethasone (Sigma D-4902)
Insulin (Bovine; Sigma I-5500)
MEM Sodium Pyruvate (100mM; GibcoBRL Cat#11360-070)
Pen/Strep/Glutamine (100x P/S/G; GibcoBRL Cat#10378-016)
SOLUTIONS
10% Calf Serum/DMEM
60mL Calf Serum
6mL 100mM MEM Sodium Pyruvate
6mL 100x P/S/G
500mL DMEM
10% FBS/DMEM
60mL Fetal Bovine Serum (Filter Sterilized)
6mL 100mM MEM Sodium Pyruvate
6mL 100x P/S/G
500mL DMEM
IBMX Solution (make fresh)
a)Dissolve IBMX in a solution made of 0.5N KOH to a final concentration of 0.0115g/mL.
b)Filter sterilize through a 0.22 mm syringe filter.
Insulin Stock Solution
167 uM (1mg/mL) in 0.02M HCl
Filter sterilized through 0.22 mm filter
Can store at -20C for long term, 4C short term.
Dexamethasone Stock Solutions
Freezer Stock: 10mM of Dex in 100% ethanol (store at -20C)
Working Stock: Dilute Freezer stock to 1mM in PBS
Filter sterilize and store at 4C.
MDI Induction Media (10mL/10cm plate; 5mL/6cm plate)
To required volume of 10% FBS/DMEM add:
1:100 IBMX
1:1000 Insulin
1:1000 Dexamethasone working stock
Insulin Media (10mL/10cm plate; 5mL/6cm plate)
To required volume of 10% FBS/DMEM add:
1:1000 Insulin
METHOD
Preadipocyte maintenance and passage:
We plate the cells in 10% CS/DMEM on treated polystyrene culture dishes from Corning (Cat#430167) and incubate them at 37C in 10% CO2. It is important to feed the preadipocytes every couple of days and avoid letting them get too confluent (>70%), if you want to continue to passage them and differentiate them at a later date. So, take care to split them appropriately. They can be split as far as 1:15, though we usually do 1:10 or less depending on need.
Adipocyte Differentiation Protocol:
1. Grow preadipocytes to confluency in 10% calf serum/DMEM
2. Two days post confluency (DAY 0) stimulate the cells with MDI induction media. You will notice a distinct change in the morphology of the cells (become more spindly) in the next 2 days.
3. Two days after MDI (DAY 2) change the media to Insulin Media. The media will begin to get more viscous as free fatty acids are produced by the cells and secreted into the media.
4. Two days later (DAY 4) change media to 10% FBS/DMEM. Feed cells with 10%
FBS/DMEM every two days. Full differentiation is usually achieved by DAY 8.。