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An Improved Quantitative Assay for Chemokinesis in Tetrahymena
Uffe Koppelhus, Per Hellung-Larsen and Vagn Leick
Vol. 187, No. 1 (Aug., 1994), pp. 8-15
Published by: The University of Chicago Press
Stable URL: http://www.jstor.org/stable/1542160
Page Count: 8
You can always find the topics here!Topics: Starvation, Amino acids, Chemical suspensions, Peptones, Insulin, Chemotactic factors, Fibroblast growth factors, Cultured cells, Hormones, Swimming
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This paper presents a quantitative and sensitive assay for the measurement of chemosensory behavior in Tetrahymena. The two-phase assay is easy to perform in large quantities, so a variety of compounds can be screened under comparable conditions. A suspension of 2× 105 cells ml-1 (the upper phase) is starved for 20-40 h and then gently placed on top of a 5% solution of Metrizamide (the lower phase) in a disposable microcuvette. The optical density of the lower phase is monitored at 600 nm with an automated spectrophotometer at selected time points. Optimum sensitivity of the assay is achieved when the cells slowly but continuously enter the lower phase, so that about 5% of them will be in the lower phase within 30 min. Optimal chemosensory responses occurred in Tetrahymena thermophila at about 25°C. The response was delayed at 15°C and markedly reduced at 35°C. The data suggest three bases for quantifying the response in the assay: (1) initial slope of the absorbance versus time; (2) final maximal absorbance within the time period of measurement; and (3) signal-to-noise ratio (S/N) at a fixed time. We have quantified-in terms of S/N-the chemosensory responses in Tetrahymena for the following compounds: β-endorphin, fibroblast growth factor, insulin, and platelet-derived growth factor (PDGF); these substances were active in nanomolar concentrations, and the maximal S/N was between 3 and 5.1. Acetylcholine was active only in millimolar concentrations; maximal S/N was 4.1 at 1 mM. Glutamic acid, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine were active in millimolar concentrations, with S/N between 1.5 and 2.5. We found S/N equal to 9.6 for a mixture of amino acids (1 mM of each amino acid). Furthermore, if PDGF was added to the mixture of amino acids, the S/N increased to 11.3. Proteose peptone at a concentration of 1 mg ml-1 produced a strong response, with S/N equal to 17.5. The assay is also suitable for the detection and quantification of repellents. We found that 20 mM KCl, 1 mM 4-nitroaniline (4-NA), and 100 μM 2,4-dinitroaniline (2,4 DNA) each almost completely prevented the cells from migrating into the lower phase. The minimum concentrations of significant repellent effect using these substances were 500 μM, 1 μM, and 50 nM, respectively.
Biological Bulletin © 1994 Marine Biological Laboratory