Novel flow cytometry compensation standards: internally stained fluorescent microspheres with matched emission spectra and long-term stability.

In flow cytometry, the emission spectral overlap of fluorescein and R-phycoerythrin is usually corrected by electronic color compensation using microspheres surface labeled with the same fluorochromes. However, the inherent chemical instability of these fluorochromes may cause inaccurate compensation. To overcome these problems, Compen-Flow beads, a new type of compensation standards were developed. The CompenFlow beads are a set of 6.0-microm-diameter polystyrene microspheres that are internally stained with selected BODIPY dye combinations. When excited by the 488-nm argon laser line, these beads show a nearly perfect emission spectral match to fluorescein-stained, R-phycoerythrin-stained and unstained lymphocytes, respectively. Moreover, since the dye molecules are oil soluble, they are contained inside the microsphere matrix instead of merely on the surface; thus, the molecules are shielded from environmental factors that could affect an exposed fluorochrome. Our results show a stable fluorescence spectral profile and constant intensity for at least 2 years stored either refrigerated or at room temperature.