164
edits
Changes
added SOP requirements
__NOTOC__
==SOP==
*[[SOP- Biosafety Cabinets]]
*[[Sop- Compressed Gases]]
*[[SOP- Vacuum Pumps]]
==Materials==
*'''DMEM''' (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)
*'''NCS''' (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store at -20)
*'''PSG''' (100X Invitrogen Cat# 100378-016)
*'''Insulin''' (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid (28.7μl/50ml) as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20*'''Dexamethasone''' (Sigma D-1756). Dissolve in ethanol as a 2.5 mM stock(0.049g/50ml). Aliquot into 1.5 tubes and store at -20.*'''MIX''' (Sigma I-5879). Dissolve Add 2.78g/50mL KOH (MIX and 0.98g/50mLKOH and bring up to 50 mL (250 mM stock). Aliquot into 1.5 mL tubes and store at -20
*'''Fibroblast Media''' (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)
*'''Adipocyte Media''' (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)*'''Differentiation DMI Media I''' (Adipocyte Media + Insulin (1000X dilution), Dexamethasone (10 000X dilution) and MIX (500X dilution); Sterile filter into new bottle after additions*'''Differentiation Insulin Media II''' (Adipocyte Media + Insulin (1000X dilution); Sterile filter into new bottle after adding insulin) [[File:3T3-L1_Adipocyte_Differentiation_Schematic.png]]
==Fibroblast Culture==
*Cells can be grown at 37C + 58% CO2, and split 10-20X. Typically healthy fibroblasts will repopulate in 2-3 days after a 10X dilution.
*Split cells when at about 80% confluence. Confluence initiates the differentiation program, so if cells reach confluence, you will need to thaw another DMSO stock and start over. Splitting cells will normally not reverse this
*When splitting cells, wash cells 2x with sterile PBS (-/-) then add 0.05% trypsin and sit at room temperature for 2-5 min. Be careful not to over-trypsinize cells
*Cells can normally be passaged up to about passage # 25 without problems.
*Plate cells out in whichever format you require your adipocytes in (ie i.e. 12 well plates or 15 cm dishes)
==Differentiation of Fibroblasts to Adipocytes==
