3T3-L1细胞诱导分化

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.