Lab Protocols

Procedure Day 0 cell culture: Grow 3T3-L1 cells in high glucose DMEM + 10% Calf Serum + P/S/F in 6-well plates. After cell have reached complete confluent, replace growth media with stimulation media containing: High glucose DMEM: 10% FCS, P/S/F 1 µl / ml of Insulin (10 mg/ml, 1000x in HEPES, final 10 µg/ml) 1 µl / ml IBMX (3-isobutyl-1-methylxanthine, 500 mM stock, 1000x), final 0.5 mM 0.1 µl / ml Dexamethasone (10000X in ethanol, final 1 µM). Day 3 Replace with fresh media containing Insulin only, no IBMX, no Dexamethasone.  Change the media on day 6.  Cells will be differentiated on day 7-9. Day 6 Cells will be cultured in serum-free DMEM for 2 hr ± treatments Incubate cells  in 1 ml of KRH with 100 nM insulin for 20 min ± treatments. Add 1 µCi of 2-deoxy-D-[2,6-3H]-glucose (50 µl in volume) to each well for 10 min. Wash cells with KRH buffer 3X. Lyse cells in 1 ml of RIPA buffer.  Count 300 µl. KRH buffer: 25 mM HEPES-NaOH [pH 7.4] 1...

Procedure describing a suggested protocol for sarcomeric actinin, ANP, ER and ERb fluorescent staining of cardiomyocytes View a pdf version of this page   Anti-sarcomeric – α actinin and ANP Staining Fix cells as usual in 3.7% paraformaldehyde in PBS. Rinse cells twice with PBS after fixation. Permeabilize and block in 2% FBS / 2% BSA in PBS with 0.1% NP40 for 45 min. Treat cells with antibodies for α-actinin eg.  alpha sarcomeric actin antibody [alpha Sr-1] (ab28052)  and ANP diluted in the blocking buffer as above for 1 hour. Wash x3 in PBS for 5 min each. Add secondary ab in the same blocking buffer (for green ab: CY2 or fluoroscein labeled abs, use 1/200 dilution of secondary ab and for CY3 or Rhodamine, use 1/1000 dilution for 45 min (though these will require some optimization) Wash in PBS x3 Add DAPI solution to coverslips for 15 min. Wash x2 in PBS. Add mounting solution and coverslip. Image cells within 2 days. The α-actinin stain is stable for quite so...