Difference between revisions of "Differentiation of 3T3-L1 Cells"

Revision as of 15:04, 18 March 2018

DMEM (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)
FBS (we test several batches from several sources for differentiation and insulin stimulated glucose uptake then order large quantities of that particular lot. Currently we are using Sigma cat# F2442, lot# 072K8403. Aliquot and store in 50 mL tubes 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)
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). 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)
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)

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
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 (i.e. 12 well plates or 15 cm dishes)

Let cells grow 2-3 days after confluence (typically 8-10 days after a 10X passage)
Add Differentiation Media I and incubate 3-4 days. Cells should shrink after day 1 then start to round up and by *day 3 should begin to accumulate lipid (the media should change color as well)
After 3-4 days, add Differentiation Media II and incubate 2-3 days (for a total of 6 days).
Change media to Adipocyte media and change every 2-3 days. Insulin responsiveness (as measured by glucose uptake) increases to a maximum at about 10 days post-differentiation.
Good cells should be homogeneous and have >10 fold insulin stimulated glucose uptake.
If adipocytes need to be trypsinized for replating or electroporation this is best done before the fourth day after changing to Adipocyte media. Use 0.25% trypsin. After that, cells do not trypsinize well.

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 *'''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).  Add 2.78g MIX and 0.98g KOH and bring up to 50 mL.   Aliquot into 1.5 mL tubes and store at -20
+*'''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)
 *'''Adipocyte Media''' (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)