Direct and highly sensitive measurement of fluorescent molecules in bulk solutions using flow cytometry.

ABSTRACT

Utilizing flow cytometry to monitor progress of bulk biochemical reactions and concentration of chemical species normally relies on the utilization of cells carrying intrinsic fluorescence or modified beads. We present a method for a simple measurement of the fluorescent marker molecule fluorescein and GFPuv in bulk solutions with high sensitivity using a CytoFLEX flow cytometer and without the need for modified beads. Polystyrene beads were used to trigger measurements based on their high scatter signal, to detect the fluorescence signal from two different fluorophores present in the sample solution. We report sensitivities of 33 pg/mL for fluorescein and 50 ng/mL for GFPuv. This method is comparable in sensitivity to a typical spectrometric fluorescence assay tested with fluorescein, and approximately ten times more sensitive for the measurement of GFPuv. PEG was added to the sample at a low concentration of 0.001% (w/v) to block unspecific GFPuv binding to the beads. The method was further applied to measure the GFPuv concentration in crude cell lysate samples used for cell free protein expression. An advantage of this method over spectrometric assays is the ability to differentiate signal subpopulations in the sample based on their individual fluorescence intensities.