Endy:FACS calibration with beads

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The Default Bead

SPHERO Rainbow Calibration Particles from Spherotech:

Cat.#: RCP-60-5



Note: As a test, beads were diluted on 12.15.05 and stored at -4C in an opaque container. The same stock of beads were freshly diluted on 01.16.06 and both tubes were compared by FACS. To the accuracy of the instrument, the measurements were identical (+/- 2%). -Jkm

FlowJo Calibration

In FlowJo, select the bead sample to calibrate against. Then choose Platform->Define Calibration. Select the parameter to calibrate, and use the beads as your reference sample. I (Jkm) use as reference values:

Peak  GFP    RFP
1.    10.3    31.1
2.    30.2    70.4
3.    83.7   224
4.   315     885
5.  1092    2922

These numbers are arbitrary - they simply are the values that I measured for one specific date. Every other measurement, then, is calibrated against that day.

Occasionally, these peaks are difficult to distinguish (if the setting are extremely high or low, peaks will not be apparent). It can become a judgment call to decide which measured peak corresponds to which calibration peak.

Uncertainty in Calibration

  • Qualitative observations from Josh (all numbers rough)
      • Same as above, only using a single point calibration.
    • Uncertainty when calibrating different machines hasn’t been evaluated.
  • Bead quality issues:


Bead Suppliers:


OWW Contacts:

Caitlin Conboy, Josh Michener, Francois St.Pierre, etc.

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