SpheroTechnical Notes #17 - Determination of a Flow Cytometer's Sensitivity Using Detection Efficiency (Q) and the Background Light Level (B)

In order to obtain accurate and scientifically sound flow cytometric data it is critical to use standardization techniques and a robust and reliable instrument. The standardization protocol should address specific operational parameters to determine the performance of the instrument at any point in time. The flow cytometer's optic, fluidic and electronics design should be simplistic while still being sensitive and effective.

Experimental Procedure
One major parameter to be included into the standardization protocol is a test for sensitivity. Sensitivity is an important parameter since it defines the ability to detect particles above background. However, sensitivity determination should also include resolution, the ability to resolve dim particles. This can be measured using Spherotech Rainbow Calibration Particles with eight intensities, Cat. No. RCP-30-5A (Rainbow Calibration Particles, 8 peaks, 1E7/mL, 3.0-3.4 um, 5 mL). Since each intensity of the RCP-30-5A has been calibrated to the molecules of equivalent fluorophores (MEF) it can be used to quantitate sensitivity. Figure 1 is the histogram of the RCP-30-5A on Stratedigm's S1000 cutting-edge flow cytometer in the PE channel. See http://stratedigm.com/instrumentation for more information on Stratedigm flow cytometers.

A flow cytometer's sensitivity can be described as the detection efficiency (Q) and the background light level (B). The detection efficiency is how well light is collected in the cytometer, while the background light level shows how much noise is created by the instrument in the background. In order to determine the efficiency (Q) and the background light level (B) the MEF values of blank beads and another dim population must be known. These values can be obtained using the Spherotech PMT QC Template.
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SpheroTechnical Note #17 will provide the following information:
  • How the the Spherotech 8 peak Rainbow Calibration Particles (Cat. No. RCP-30-5A) provides robust and reliable data when a standardization protocol is successfully adopted and implemented.
  • A introduction on using the Rainbow Calibration Beadsfor the calculation of a flow cytometer's sensitivity by measuring the Detection Efficiency (Q) and the Background Light Level (B)
  • Why a flow cytometer standardization protocol should include the statistical analyses of sensitivity measurements to promote innovative, scientifically sound experimental results.