Differentiation of 3T3-L1 Cells: Difference between revisions
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__NOTOC__ | |||
==SOP== | |||
*[[SOP- Biosafety Cabinets]] | |||
*[[Sop- Compressed Gases]] | |||
*[[SOP- Vacuum Pumps]] | |||
==Materials== | ==Materials== | ||
*'''DMEM''' (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092) | *'''DMEM''' (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092) | ||
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*'''NCS''' (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store at -20) | *'''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) | *'''PSG''' (100X Invitrogen Cat# 100378-016) | ||
*'''Insulin''' (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20 | *'''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. Aliquot into 1.5 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). | *'''MIX''' (Sigma I-5879). Add 2.78g MIX and 0.98g KOH 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) | *'''Fibroblast Media''' (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle) | ||
*'''Adipocyte Media''' (DMEM + 10 % FBS + 1X PSG; Sterile filter into new bottle) | *'''Adipocyte Media''' (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle) | ||
*''' | *'''DMI Media''' (Adipocyte Media + Insulin (1000X dilution), Dexamethasone (10 000X dilution) and MIX (500X dilution); Sterile filter into new bottle after additions | ||
*''' | *'''Insulin Media''' (Adipocyte Media + Insulin (1000X dilution); Sterile filter into new bottle after adding insulin) | ||
[[File:3T3-L1_Adipocyte_Differentiation_Schematic.png]] | |||
==Fibroblast Culture== | ==Fibroblast Culture== | ||
*Cells can be grown at 37C + | *Cells can be grown at 37C + 8% 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 | *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 | *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. | *Cells can normally be passaged up to about passage # 25 without problems. | ||
*Plate cells out in whichever format you require your adipocytes in ( | *Plate cells out in whichever format you require your adipocytes in (i.e. 12 well plates or 15 cm dishes) | ||
==Differentiation of Fibroblasts to Adipocytes== | ==Differentiation of Fibroblasts to Adipocytes== | ||
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==Reference== | ==Reference== | ||
PMID 16472699 | |||
[[ Category:Cell Culture ]] | [[ Category:Cell Culture ]] | ||