Luciferase assay for determining the physical condition of cells

• Main Authors: DAVID PETER SOWTER, SHARON PATRICIA MARY CROUCH, KEVIN JOHN SLATER
• Format: Patent
• Language: English
• Published: 16.09.1998
• Edition: 6
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; ENZYMOLOGY; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIRCHEMICAL OR PHYSICAL PROPERTIES; MEASURING; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; METALLURGY; MICROBIOLOGY; MUTATION OR GENETIC ENGINEERING; PHYSICS; PROCESSES OF PREPARING SUCH COMPOSITIONS; SPIRITS; TECHNICAL SUBJECTS COVERED BY FORMER USPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ARTCOLLECTIONS [XRACs] AND DIGESTS; TESTING; VINEGAR; WINE

A method for determining the physical condition of cells comprises (a) adding an agent to cells which releases adenylate nucleotides therefrom, for example an anionic or non-ionic detergent; (b) adding luciferin and luciferase and accordingly determining the ATP concentration by measuring the intensity of light emitted; (c) when ATP levels are substantially constant, adding a reagent which converts ADP to ATP, preferably a mixture of phosphoenol kinase and pyruvate kinase, and measuring the light emitted to determine the ADP concentration; (d) determining the ADP:ATP ratio. The method can be used to distinguish between, apoptotic cells, necrotic cells, proliferating cells and growth arrested cells. The magnitude of the ADP:ATP ratio will be necrotic cells > apoptotic cells > proliferating cells > growth arrested cells. The application of the method to mammalian cell lines and a kit for carrying out the method are described. The anti-cancer drugs camptothecin and etoposide are incorporated into some of the assays.