Use these beads to calibrate your flow cytometer prior to analyzing cells that express the AcGFP1 or mCherry fluorescent proteins. The AcGFP flow cytometer calibration beads allow for easy calibration of any flow cytometer with a 488-nm laser line that excites the green fluorescent proteins AcGFP1 (Aequorea coerulescens GFP) and EGFP. (The AcGFP flow cytometer calibration beads work for both AcGFP1 and EGFP because their excitation/emission spectra and brightness are almost identical.) The mCherry flow cytometer calibration beads allow for easy calibration of any flow cytometer with a 561-nm laser line that excites the red fluorescent protein, mCherry.

Each bead suspension contains six distinct populations of beads that vary in the number of attached AcGFP1 or mCherry molecules, which gives each population a distinct fluorescence intensity. The Molecular Equivalent of Soluble Fluorophore (MESF) value for each peak is determined by correlating the fluorescence intensity of each respective bead population with the amount of soluble AcGFP1 or mCherry yielding the same fluorescence intensity.

Converting fluorescence intensity readings to MESF makes it possible to estimate and compare relative expression levels between cells in the same cell population or in independent samples, and even to compare data from different instruments and experiments. Using the flow cytometer calibration Beads, you can also determine the linearity and stability of your flow cytometer’s readouts. The lowest intensity peak represents the autofluorescence signal of cells not expressing the green or red fluorescent protein; this allows you to measure the fluorescence detection threshold and the background noise level of your flow cytometer. The five remaining peaks are evenly distributed over the remaining scale of the fluorescence detection channel.