Conversion of logarithmic channel numbers into relative linear fluorescence intensity

Abstract

We describe a simple, reproducible, and generally applicable method to assess the performance of log amplifiers by using a fluorescent sample that provides multiple peaks of different intensities. The channel differences between multiple peaks are used to evaluate the logarithmic behavior of the fluorescence signal amplifier on the flow cytometer. A calibration curve can be created to correct the channel numbers for deviations from true logarithmic behavior and then convert data into relative linear intensities. By using these linear fluorescent intensities, we compared the capacity of different antisera against HIV-1 (human immunodeficiency virus type 1) peptides to inhibit the binding of HIV-1 to CEM, a CD4-positive T-cell line. A wide range of applications for this calibration procedure can be envisioned and the method is valuable for monitoring instrument performance over time.