Endy:FACS calibration with beads
The fluorescence level measured by flow cytometry is in arbitrary units and entirely dependent on the instrument settings used in taking the measurement. Even when consistent settings are used, variability in instrument performance makes comparison between data sets acquired on different days uncertain unless the instrument is calibrated.
Running a sample of calibration beads as a control in flow experiments allows normalization of multiple data sets for comparison (even if acquired with different pmt settings or on different cytometers). Additionally, the level of bead fluorescence can be experimentally correlated to absolute units, such as Molecules of Equivalent GFP (MEG). This allows users to approximate copies per cell of GFP from flow data, based on earlier quantitative western blot characterization.
Note: Any beads can be used. As long as they’re run against The Default Bead the data can be normalized. As a general rule, beads with multiple intensity peaks will give better calibration (see uncertainty below). Beads with a rainbow of fluorophores are most versatile.
The Default Bead
SPHERO Rainbow Calibration Particles from Spherotech:
6.0- 6.4 um beads. (Size recommended by Glenn Paradis at the MIT flow facility. Not sure if there’s a strong rationale for this.)
The cost of these beads is $235.00 per 5 mL, or < $0.05 per experiment, particularly if you store and reuse diluted beads, as recommended.
Uncertainty in Calibration
- Josh’s observations (Josh, feel free to elaborate on this or correct me if I’m wrong. ~ cmc 11:42, 10 Nov 2005 (EST))
- Uncertainty <5% when calibrating data sets from the same instrument with the same settings.
- Uncertainty ~10% when calibrating data acquired on the same instrument with variable pmt settings.
- Uncertainty ~20% when calibrating between different pmt settings if only using one calibration peak.
- Uncertainty when calibrating different machines hasn’t been evaluated.
- Bead quality issues:
- Bead lifetime. (“very stable under proper storage conditions” means…?)
- Lot to lot bead variability. Could retain one master lot to calibrate all new lots against. This comes back to bead lifetime eventually. Certainly any new lot of beads should be run against the previous lot.