In situ assay of hormone-stimulated adenylyl cyclase in 96-well microtitration plates: an aide to rapid identification of transformed cell clones.
An in situ assay able to detect hormonally stimulated or inhibited adenylyl cyclase (AC) activity on as few as 5,000 cells/well has been developed. In addition the assay monitors phosphatase activity which serves as a marker for cell density. Cells are plated in replicate wells at least one day before the assay, and the medium containing AC reagents, an ATP regenerating system, a PDE inhibitor, additives that regulate receptors and/or G proteins, and 5 mM p-nitrophenyl phosphate (pNPP), plus Tris buffer to pH 7.5 is added. The hypotonic medium causes permeabilization of cells without massive lysis. After stopping the reaction with 100 microliters of a solution with ATP, cAMP and SDS, the color indicating phosphatase activity is quantified by an ELISA reader, and AC activity measured by standard methods. At proper cell density (pNPP hydrolysis) the assay shows proportionality up to 2 hours. The assay is particularly useful in transfection experiments. As few as 50,000 cells can be plated and identified as receptor "positive" or receptor "negative". The assay was key to our cloning of the V2 AVP receptor. The assay accelerated the preparation of stable cell lines with LH, FSH, adrenergic and serotonin 1D beta/1B and 1E receptors. It should also be useful in studies in which the transfected cDNA encodes the adenylyl cyclase proper.
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
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Themmen
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Hinrichs
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Birnbaumer
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002461:Cell Line, Transformed; D002999:Clone Cells; D005821:Genetic Techniques; D051379:Mice; D008846:Micromanipulation; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000502:physiology*; D011974:Receptors, LH / Q000378:metabolism; D011985:Receptors, Serotonin / Q000378:metabolism; D013997:Time Factors; D017186:Titrimetry / Q000295:instrumentation; D014162:Transfection
D011943:Receptors, Adrenergic, beta; D011956:Receptors, Cell Surface; D011974:Receptors, LH; D011985:Receptors, Serotonin; D000262:Adenylyl Cyclases