Sheath fluid control to permit stable flow in rapid mix flow cytometry.

BACKGROUND

Flow cytometry is a potentially powerful tool to analyze the kinetics of ligand binding, cell response and molecular assembly. The difficulty in adding reactant to cells, achieving adequate mixing, delivering those cells to the laser focal point and establishing stable flow, has historically limited flow cytometry to systems with reactions times longer than 5 s. With the advent of automated syringes and flow injection methods, sample injection times shorter than 1 s have become routine. However, an inherent problem in acquiring time courses starting under 1 s is that rapid sample introduction through the flow tip to the detection point perturbs laminar flow. The purpose of this work was to determine if stable flow could be reestablished more quickly if the sheath flow was reduced during sample introduction, returning to normal sheath and sample rates afterward.

METHODS

We used programmable syringes and valves to control sample mixing as well as sheath and sample delivery through the flow tip to the detection point for stream-in-air detection. Stable flow was monitored by mean particle fluorescence during sample introduction.

RESULTS

With no sheath reduction, stable flow recovered after more than 1 s. By reducing sheath flow during the short period (300 msec) of sample mixing and delivery, stable laminar flow recovered within 200 msec.

CONCLUSIONS

This use of automated syringes to control both sheath and sample flow provides a potential for robust sample handling applicable to kinetic as well as high throughput flow cytometric analysis.