A RAPID MIX FLOW CYTOMETER WITH SUBSECOND KINETIC RESOLUTION

Kinetic approaches are valuable tools for mechanistic studies of cell function, Flow cytometry is well suited to make sensitive kinetic meas urements, but the time required to deliver mixed samples to the point of measurement (10-20 s in a conventional cytometer) limits analysis o f rapidly occurring events, To address this limitation, we adapted a s yringe-based stopped-now rapid mixing device to a modified commercial now cytometer to achieve mixing and measurement of sample in under 1 s , Because such screw-driven mixers are designed to deliver fluid at ra tes of microliters per millisecond and cytometers accept samples at mi croliters per second, the syringe mixer was modified with a screw to a llow sample delivery at rates as low as 1.8 mu l/s. A custom-made nozz le holder featuring a fast-acting three-way sample delivery valve and a 1.5-mu l dead volume was designed for a Becton Dickinson FAGS stream -in-air flow nozzle, Syringe motors and valves are computer controlled , as is the start signal for an adjustable time ramp. A stable sample stream can be established within the sheath stream in less than 1 s, e nabling fluorescence measurements of microspheres with coefficients of variation of similar to 5%, Light scatter gating to select particles in the center of the laser beam enables fluorescence measurements at t imes of under 300 ms. Efficient mixing of reagents is demonstrated by the iodide quenching of microspheres surface labeled with fluorescein isothiocyanate (FITC), The instrument is capable of quantitatively pro portioning cells and reagent, thereby allowing precise control of reag ent concentration and dilution, Rapid kinetic measurements of intact c ells are demonstrated by PITC-formyl peptide binding to cell surface r eceptors. (C) 1995 Wiley-Liss, Inc.