A rapid mix flow cytometer with subsecond kinetic resolution

Abstract

Kinetic approaches are valuable tools for mechanistic studies of cell function. Flow cytometry is well suited to make sensitive kinetic measurements, but the time required to deliver mixed samples to the point of measurement (10-20 s in a conventional cytometer) limits analysis of rapidly occurring events. To address this limitation, we adapted a syringe-based stopped-flow rapid mixing device to a modified commercial flow cytometer to achieve mixing and measurement of sample in under 1 s. Because such screw-driven mixers are designed to deliver fluid at rates of microliters per millisecond and cytometers accept samples at microliters per second, the syringe mixer was modified with a screw to allow sample delivery at rates as low as 1.8 microliters/s. A custom-made nozzle holder featuring a fast-acting three-way sample delivery valve and a 1.5- microliters dead volume was designed for a Becton Dickinson FACS 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, enabling fluorescence measurements of microspheres with coefficients of variation of approximately 5%. Light scatter gating to select particles in the center of the laser beam enables fluorescence measurements at times 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 proportioning cells and reagent, thereby allowing precise control of reagent concentration and dilution. Rapid kinetic measurements of intact cells are demonstrated by FITC-formyl peptide binding to cell surface receptors.