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.
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