Analysis of cells via flow cytometry requires calibration of the instrument with the fluorophore used to label the cells of interest. Commercially-available calibration beads are an indispensable tool when preparing flow cytometers for experiments using fluorescent dyes (e.g. FITC); however, due to the different spectral characteristics of fluorescent proteins versus fluorescent dyes, existing fluorescent dye beads are not suitable for instrument calibration if the cells being analyzed express fluorescent proteins. Therefore, we developed calibration beads labeled with either the red fluorescent protein mCherry or the green fluorescent protein AcGFP, which has spectral properties almost identical to EGFP.
Beads with a very low size deviation (CV 2.5–3%) were used to create distinct fluorescent bead populations by covalently linking specific amounts of the respective fluorescent proteins. The low size deviation of the beads, in conjunction with a very controlled labeling method, allowed us to create six bead populations with distinct fluorescent intensities for each of the two fluorescent proteins.
Here, we show that the fluorescent protein Flow Cytometer Calibration Beads are easy to use and that they perform equally well on a variety of flow cytometer platforms. We also present data showing that the mean fluorescence intensity of the beads and the calculated number of fluorescent proteins on each respective bead population are distinct from each other and in a linear correlation. We also provide supporting data showing the signal stability of the calibration beads under different buffer and fixative conditions, as well as at different flow rates. The data show that these calibration beads are a very useful tool, enabling fast and reliable calibration of flow cytometers prior to analysis of cells expressing the corresponding fluorescent protein.