ABSTRACT
Microscope-based cytometry provides a powerful means to study cells in high throughput. Here we present a set of refined methods for making sensitive measurements of large numbers of individual Saccharomyces cerevisiae cells over time. The set consists of relatively simple 'wet' methods, microscope procedures, open-source software tools and statistical routines. This combination is very sensitive, allowing detection and measurement of fewer than 350 fluorescent protein molecules per living yeast cell. These methods enabled new protocols, including 'snapshot' protocols to calculate rates of maturation and degradation of molecular species, including a GFP derivative and a native mRNA, in unperturbed, exponentially growing yeast cells. Owing to their sensitivity, accuracy and ability to track changes in individual cells over time, these microscope methods may complement flow-cytometric measurements for studies of the quantitative physiology of cellular systems.
Subject(s)
Image Cytometry/methods , Microscopy, Fluorescence/methods , Proteins/analysis , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae/chemistry , Flow Cytometry , Fluorescence , Green Fluorescent Proteins/analysis , HL-60 Cells , Humans , Proteins/metabolism , RNA Stability , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/metabolism , Sensitivity and Specificity , Time FactorsABSTRACT
A set of haptens structurally resembling the herbicide imazethapyr (PURSUIT) was synthesized and used to derive monoclonal antibodies (MAbs) and direct and indirect competition enzyme immunoassays (EIAs) which could detect imazethapyr, imazaquin (SCEPTER), imazapic (CADRE), and imazamox (RAPTOR) in the 3-30 ng/mL (parts per billion) range, and imazapyr (ARSENAL) and imazamethabenz-methyl (ASSERT) in the 300-500 ppb range. Two MAbs, 3A2 and 3A5, had affinities of 10-75 nM for imazethapyr. MAbs 1A5, 1D2, and 3A5 were specific for the S isomers of the herbicides. Some MAbs were stable in solutions containing up to 15% methanol and 5% acetonitrile in indirect EIAs. Plates coated with hapten conjugates for indirect EIA could be stored frozen. Selectivity for the imidazolinones by some MAbs varied with different coating conjugates. These MAbs and haptens should prove useful in immunochemical analysis and residue recovery methods for imazethapyr and other imidazolinone herbicides.
