Flow cytometric analysis of mature adipocytes

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 μm 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 μm diameter orifice; (3) using a short, 300 μm 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.