Difference between revisions of "Differentiation of 3T3-L1 Cells"
From Bridges Lab Protocols
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__NOTOC__ | __NOTOC__ | ||
| + | ==SOP== | ||
| + | *[[SOP- Biosafety Cabinets]] | ||
| + | *[[Sop- Compressed Gases]] | ||
| + | *[[SOP- Vacuum Pumps]] | ||
==Materials== | ==Materials== | ||
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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 | *'''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. | *'''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 % L1-FBS + 1X PSG; Sterile filter into new bottle) | *'''Adipocyte Media''' (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle) | ||
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*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. | *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 (i.e. 12 well plates or 15 cm dishes) | *Plate cells out in whichever format you require your adipocytes in (i.e. 12 well plates or 15 cm dishes) | ||
Latest revision as of 21:56, 4 June 2020
SOP
Materials
- 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)
File:3T3-L1 Adipocyte Differentiation Schematic.png
Fibroblast Culture
- 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)
Differentiation of Fibroblasts to Adipocytes
- 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.
