Lab 7 - Calibration and Standardization in Flow Cytometry

John Nolan

 

The calibration and standardization of instruments, reagents, and assays is essential for quantitative flow cytometry. This lab will provide a hands-on introduction to the basic procedures required to calibrate and standardize flow cytometers and fluorescence-labeled antibodies, and to make quantitative measurements of antigen-bearing particles. Students will learn to evaluate the performance of a flow cytometer, including measurement of background signals and sources, and fluorescence response. Fluorescence antibodies will be characterized with respect to fluorophore to protein ratio and relative quantum yield. Finally, the calibrated instrument and reagents will be used to make quantitative measurements on the number of antibody binding sites on individual particles. The assumptions and uncertainties in these measurements will be discussed to emphasize the limits to absolute quantification in flow ytometry.