Flow cytometry is an excellent method for studying the physiological function in adipocytes because their response to hormones, especially insulin, varies with cell maturity and therefore size. Adipocytes present a unique technical challenge. A freshly prepared adipocyte suspension contains cells and fat droplets ranging from 10 to >120 pm in diameter. Stored fat occupies 90-98% of the cell volume, making it difficult to distinguish cells from fat droplets. Other difficulties include buoyancy, large size, fragility, and tendency to aggregate and clog the sample tube and nozzle.These obstacles were overcome by 1) maintaining the sample, sample line, sheath fluid, reservoir, and nozzle assembly at 37°C; 2) using a 200 pm diameter orifice; 3) using a short, 300 pm inside diameter Teflon sample delivery line; 4) injecting the sample at constant flow rate into the sheath fluid at low pressure; and 5) using the pH-sensitive vital stain, biscarboxyethylcarboxyfluorescein (BCECF) to distinguish cells from fat droplets.Stained cells are brightly fluorescent when excited at 488 nm. Because fat droplets do not fluoresce, they can be distinguished from fat cells by gating on the BCECF emission. The cytosolic pH of intact, viable, mature adipocytes was derived from the ratio of the fluorescent emission intensities at 520 and 620 nm and was estimated to be 7.2. Unlike BCECF, several other useful fluorescent probes of cell function, e.g., the intracellular calcium indicator, indo-1, label both fat cells and fat droplets. Using a dual-excitation-laser flow cytometer and by gating on the BCECF emission in an adipocyte suspension labeled with both BCECF and indo-1, one can exclusively monitor cell-associated indo-1 fluorescence in order to assess unambiguously intracellular calcium concentration in viable, mature adipocytes.Key terms: Fat cell, BCECF, intracellular pH, fluorescence, The adipocyte is an extraordinarily hormone-sensitive cell that possesses receptors for a wide variety of hormones, including insulin, growth hormone, adrenergic agonists, and many growth factors (13). The adipocyte has a n unparalleled sensitivity to insulin, producing a more than tenfold stimulation of glucose transport in adipocytes compared with 40-100% stimulation in muscle cells (231, making it the cell of choice for studying insulin action and insulin resistance (13,161.Although fluorescent probes are particularly effcacious for evaluating function in living cells (1,24), available data on fluorescence analysis of adipocytes are scarce. Adipocytes are fragile and possess one un-
