Cellometer auto t4 counter

The murine non-differentiated brown adipocyte cell line [18 (link)] (kindly provided by Dr A. Whittle, Institute of Metabolic Science, University of Cambridge, UK) was cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 25 mM glucose, 10% fetal calf serum (FCS), 1% glutamine, 1% penicillin and 1% streptomycin in a humidified atmosphere at 37°C and 5% CO₂. Differentiation of the brown adipocyte cell line into mature brown adipocytes was conducted as described [18 (link),19 (link)]. The density of non-differentiated brown adipocytes depending on the further experimentation was determined by the Cellometer Auto T4 counter (Nexcelom Bioscience).

A total of 63 cell lines available at the NCFAD were seeded from frozen stocks and grown for 48 h at 37 °C and 5% CO₂, except for Trichoplusia ni cells which were grown with shaking at 27 °C for 24 h before collection. Adherent cell lines were dissociated using 0.25% Trypsin-0.1% EDTA, and cells in suspension were spun down at 4 °C for 10 min at a relative centrifugal force of 600, and re-suspended in culture media. An aliquot of cells was stained with 0.2% trypan blue and counted using a Cellometer Auto T4 counter (Nexcelom Bioscience).