Cell Treatment and Lysis

1. 3T3-L1 preadipocytes are grown in 10-cm dishes in Dubelcco's modified Eagle's-H21 medium(DMEM) supplemented with 10% calf serum. Two days postconflu-

ence, the cells are differentiated into adipocytes by replacing the media with DMEM supplemented with 10% fetal bovine serum, 1 ig/mL dexamethasone, and 112 ig/mL isobutylmethylxanthine. After 3 d, cells are transferred to DMEM supplemented with 10% fetal bovine serum. Media should be replaced at least once a week. The cells can be used for the PI3K assays 7-35 d postdifferentiation. Use a 10-cm plate of differentiated 3T3-L1 adipocytes for each treatment condition.

2. Serum-deprive the cells by washing them in PBS and then incubating them in Lei-bovitz L-15 buffer supplemented with 0.2 % BSA for 2 h at 37°C.

3. Stimulate selected plates with insulin (100 nM final concentration) for 1-3 min (see Note 6); then, wash the cells with ice cold PBS. Remove the PBS by aspiration.

4. Immediately lyse the cells by adding 1 mL of ice-cold lysis buffer, scraping the cells off the plate, transferring them to a microfuge tube, and incubating them on ice for 20 min. Centrifuge the lysate for 10 min at 4°C at top speed in a microcentrifuge (20,800g). Transfer the supernatant to new microfuge tubes. Keep the lysates on ice while performing protein assays on each sample. One can still detect insulin-stimulated PI3K activity after freezing the lysates and storing at -20°C, but activity is lost after successive freeze-thaw cycles.

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