3T3-L1 adipocyte differentiation
Reagents:
- Insulin -5000x (800 uM)
- Dissolve 50 mg insulin in 10 ml of H2O
- Add 4 ul of conc. HCl
- Methylisobutylxanthine - 111 mg/10 ml = 50 mM
- Use NaOH to put it in solution (pH = 10-11)
- Dexametazone - 4.9 mg/50 ml of 100 % ethanol (250 uM)
Note: If you want to propagate this cell line and sustain its ability to differentiate, DO NOT ALLOW THESE CELLS TO BECOME CONFLUENT before splitting (less than 50% confl.). Only cells that are chosen to undergo differentiation should reach confluence.
We passage the cells up to passage 20
We get optimal differentiation results on Corning plastic (no pretreatment required). Falcon is ok, but not as good.
The protocol is from Philipp Scherer lab.
1. Add filtered “FCS” (Fetal Calf Serum) to subconfluent 3T3-L1 fibroblasts.
500 ml DME 50 ml FCS 1X L- Glutamine (5 ml from 100X stock or 200 mM) 1X Penicillin-streptomycin (5 ml from 100X stock or 10,000 units/ml)2. After 1-2 days at confluence in “FCS” split one plate with cells.
Add 3 ml of Trypsin, Wait a few minutes until cell lift, prealiquot 10 ml FCS to 5 new plates, add “FCS” (7 ml) to the cells with Trypsin. Mix by pipetting up and down, add 1-2 ml of the diluted plate to the prealiquot plates.
3. Once the rest of the plates reach confluence in “FCS” the media is changed every two days.
Day 0 : DM 1
“FCS” Insulin (5000x), 200 ul from stock 800 uM Dexametazone (1000x) i.e. 1 ml from stock 250 uM Methyl isobutylxathine (100x) i.e. 10 ml from stock 50 mM Filter using 0.22 um filter.
Day 2 : DM 2
“FCS” Insulin (5000x), 200 ul from stock 800 uM Filter using 0.22 um filter.
Day 4 : “FCS”
Day 6 : “FCS”
Day 8 : “FCS”
Day 10 : “FCS”
Day 12 : “FCS”
Day 14 : Discard