Resolution Resource

Using the Q B and S Calculator

Introduction

Typical users of Flow Cytometers want results from their assays that are binary. That is, they want an unambiguous result of positive and negative for a given feature under test. In practice, they usually get overlap of the positive and negative populations because of the resolution of the instrument and the limitations of the reporter they are using in their assay. Defining the Resolution of a Flow Cytometer is therefore a useful thing to do, not only to test for reproducibility of the instrument's setup, but also testing different setups and picking the best instrument configuration. The measurements used to define sensitivity and resolution in a Flow Cytometer are Q, B and S, where Q is defined as the Quantum Efficiency of the system, B is the Background noise and S is the Separation Parameter -the instrument's ability to resolve two dim populations (1,2,3).

Reagents

Rainbow calibration 8 peaks particles (Spherotech, cat. no. 559123), or similar beads with low intrinsic CVs

Quantum™ FITC MESF Beads kit (beads No. 2, Bangs Laboratories Inc, cat. No. 555B)

Flow Cytometer

Vortex mixer

Laboratory sonication bath.

1) Sonicate the beads

2) Mix 4 drops Rainbow beads with 4 drops Quantum beams and add 1ml BSA

3) Vortex the mixture and run on a low setting on the cytometer.

Notes:

The extra MESF beads make the system portable between different Rainbow bead stock and different types of beads, Also, we know what the fluorescence dye is on the MESF bead, it's FITC in this case.

Analyse the results:

Enter the CV, Median and SD of the Blank beads and the Dull beads. Enter the CV of the Bright beads. Finally enter the MESF value and Median of the MESF bead.

Results:

Q is the number of photoelectron equivalents per MESF molecule. The higher the better!

B is the noise measured in MESF. The lower the better!

S is the Separation parameter, a unit-less value in which the higher the number, the better the separation is!

R² is the Regression line through the data. The closer to 1 the better.

Q, B and S: Evaluating the Sensitivity of a Flow Cytometer