title: Free insulin 100nM 4h Bio1
organism: Mus musculus
description: Protocols: Cells were washed with PBS and lysed directly in cell culture well using lysis buffer provided in Quick-RNA MicroPrep Plus kit (Zymo Research). Brown adipocytes were differentiated from an immortalized brown preadipocyte mouse cell line previously described (PMID: 16679291). Preadipocytes were maintained and expanded at low confluence (<50 %) in growth media [DMEM high glucose (Gibco) supplemented with 20% fetal bovine serum (Sigma-Aldrich), 20 mM HEPES (Sigma-Aldrich), and 100 U/mL penicillin /streptomycin (Gibco)]. For differentiation, preadipocytes were washed twice with phosphate-buffered solution (PBS), dissociated with TrypLE Express, and seeded at a density of approximately 23,000 cells/cm2 in growth medium. Medium was changed after 24 hours to differentiation medium [DMEM high glucose supplemented with 10% fetal bovine serum, 20 nM insulin, 1 nM triiodothyronine (T3), and 100 U/mL penicillin/streptomycin] and cells were cultured for 2 days. Cell differentiation was then induced by addition of induction media (differentiation media supplemented with 0.125 mM indomethacin, 0.5 Î¼M dexamethasone, and 0.5 mM isobutyl methylxanthine) for two days, after which, medium was changed to differentiation medium for two additional days. Differentiated adipocytes were serum starved for 2 hours using starvation medium [DMEM low glucose (Gibco) supplemented with 2.5 mM glucose (to a final concentration of 8mM), 0.5% bovine serum albumin (BSA) (Sigma-Aldrich), and 100 U/mL penicillin /streptomycin] before treatment with Insulin-NanoRod structures or insulin diluted in starvation medium for four hours. For control sample only starvation media was used. Following treatment, cells were washed one time in PBS and harvested for RNA isolation. Total RNA was isolated from differentiated adipocytes using Quick-RNA MicroPrep Plus kit (Zymo Research) TruSeq RNA Library Prep Kit v2 Low Sample Protocol, 500ng RNA input