A simple and rapid method for determining the linearity of a flow cytometer amplification system

Abstract

We describe a simple and rapid method for determining the linearity of a flow cytometer amplification system. The method is based on a fundamental characteristic of linear amplifiers: The difference between two amplified signals increases linearly with increasing amplifier gain. Two populations of beads or cells, differing slightly in fluorescence intensity, are analyzed by the flow cytometer at increasing photomultiplier tube high-voltage settings. The distribution of the populations' mean difference versus mean position is a straight line intersecting the origin for linear amplifiers. Although some types of nonlinearities cannot be detected with this technique, deviations from linearity indicate nonlinear components in the flow cytometer amplification system. The correlation coefficient is used to quantify degree of nonlinearity. We also describe a method for amplifier nonlinearity compensation.

Nuclear genome size in Selaginella

“…or Oryza sativa L. as internal standards are approximately 1.5 times larger (0.13 pg/1C). Since the genome sizes of G. max and O. sativa are, respectively, 12.5 and 5.6 times larger than the lowest (and most reliable) published value for S. moellendorffii (Wang et al 2005), this discrepancy is presumably the result of nonlinearity-i.e., a compression artifact of linear amplification that is exasperated by larger ranges of signal intensity (Bagwell et al 1989;Doležel and Bartoš 2005). These discrepancies cannot be attributed to the secondary chemistry of S. moellendorffii and (or) nuclear autofluorescence given the data published by Wang et al, which clearly demonstrate an increase in estimated S. moellendorffii genome size with increasing internal standard size.…”

T Lymphocyte Adhesion to Human Synovial Fibroblasts. Role of Cytokines and the Interaction Between Intercellular Adhesion Molecule 1 and CD11a/CD18

“…Fluorescence intensity was measured on a 3-decade log amplifier. Linearity of the amplifier was confirmed using previously described methods (25). Mean channel fluorescence values were then converted to relative linear values, for direct comparison of intensity differences (26).…”

Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS – part III – analytical issues

“…Fixed nuclei are used as standards for DNA measurements or tools for linearity of fluorescence verification (2,3). Stabilized cells can potentially be used as controls and calibrators (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19).…”