Bright Light, No Lysis

Transcription

1 Bright Light, o Lysis By Erika Hawkins 1, M.S., James Unch 2, Ph.D., ancy Murphy 1, B.S., Jolanta Vidugiriene 1, Ph.D., Mike Scurria 2, Dieter H. Klaubert 2, Ph.D., and Keith V. Wood 1, Ph.D., 1 Promega Corporation and 2 Promega Biosciences, Inc. ViviRen TM EnduRen TM Live Cell Substrate -h ViviRen TM Substrate 3 EnduRen TM Substrate 24 CellTiter-Glo Reagent Chroma-Luc TM Red Luciferase EnduRen TM ViviRen TM Live Cell Substrate EnduRen TM ViviRen TM Live Cell Substrate Live Cell Substrate Live Cell Substrate -h 1 -h 2 A B 2 Live Cell Substrate ViviRen TM EnduRen TM Live Cell Substrates ViviRen TM Substrate 2 C ViviRen TM Substrate 3 ViviRen TM Substrate / 288 ViviRen TM EnduRen TM Substrate ViviRen TM Substrate EnduRen TM Substrate ViviRen TM Substrate EnduRen TM Substrate 1 -h EnduRen TM Substrate 24 Live Cell Substrate EnduRen TM ViviRen TM Live Cell Substrate -h -h % FBS % 17 5% 1 -h 1 2

2 A. Intracellular esterase EnduRen Live Cell Substrate H H Luciferase (Renilla, Aequorea, belia, Gaussia) H Coelenterazine-h H B. Coelenteramide-h H 2 EnduRen Live Cell Substrate H 2 ViviRen Live Cell Substrate 4945MA 1. Live Cell Substrate A. EnduRen TM Live Cell Substrate -h ViviRen TM Live Cell Substrate B. EnduRen TM ViviRen TM Live Cell Substrate ViviRen TM Substrate EnduRen TM Substrate 2 45 EnduRen TM Substrate 3 EnduRen TM Substrate 1 EnduRen TM Substrate 1 CH RAi sira 33 4 ViviRen TM EnduRen TM Substrate CH HeLa HEK293 IH/3T3 2, 3 Live Cell Substrate ATP CellTiter-Glo Reagent 1 Chroma- Luc TM Luciferase pcbr-basic Vector [ E1411] pcbr-control Vector [ E1421] CellTiter-Glo Reagent 2 EnduRen TM ViviRen TM Substrate CellTiter-Glo Reagent ATP CellTiter-Glo Reagent -h 11

3 A. B.,, [Coelenterazine] (µm),, 1 Luminescence Autoluminescence [ViviRen Substrate] (µm) C. Signal-to-Background Ratio Coelenterazine ViviRen Substrate [Substrate] (µm) 4811MA 2. in situ CH CH % FBS F12 CHA ViviRen TM Substrate B ViviRen TM Substrate n=6 2 Turner BioSystems Veritas TM Luminometer A B C,, ViviRen Substrate EnduRen Substrate Time (minutes) 3. ViviRen TM Substrate EnduRen TM Substrate 2 45 CH 6µM ViviRen TM EnduRen TM Substrate 1 2 Turner BioSystems Veritas TM Luminometer n= MA 2-12 moles 6µM -h CellTiter-Glo Reagent 7,,9 CellTiter-Glo Reagent DMEM +% 56% 4.7% In situ Live Cell Substrate Chroma-Luc TM Luciferase 611nm 488nm 5A 2 CRE SV4 Chroma-Luc TM 12

4 Renilla luminescence (% of nonspecific nucleotide sira) nM sira 25nM sira A. Relative Intensity (% of maximum) Renilla pcbr-control Vector Wavelength (nm) Time (hours post transfection) 4. EnduRen TM Substrate RAi CH sira 5nM 25nM sira sira MA B. Signal Induction Renilla luciferase ormalized luminescence EnduRen TM Substrate CRE 5 5B Chroma-Luc TM 43% 24% in situ 1 EnduRen TM ViviRen TM Live Cell Substrate Time (hours after isoproterenol & R addition) 5. Chroma-Luc TM A. Chroma- Luc TM pcbr-control Vector [ 1421] CH 2 5/6nm 6 B. Chroma-Luc TM HEK CRE PEST CL1 2 SV4 Chroma-Luc TM pcbr-control Vector [ E1421] 24 6µM EnduRen TM Substrate 2mM 2 1µM isoproterenol µm Ro Calbiochem, LaJolla, CA Turner BioSystems Veritas TM Luminometer E /6 Chroma-Luc TM 6 Chroma-Luc Calculator n= MA 13

5 1. Hawkins, E.M. et al. (22) In: Bioluminescence & Chemiluminescence Progress & Current Applications. Stanley, P.E., and Kricka, L.J., eds. World Scientific, Singapore, EnduRen Live Cell Substrate Technical Manual, #TM244, Promega 3. ViviRen Live Cell Substrate Technical Manual, #TM64, Promega EnduRen Live Cell Substrate Technical Manual, #TM244, Promega ( ViviRen Live Cell Substrate Technical Manual, #TM64, Promega ( CellTiter-Glo Luminescent Cell Viability Assay Technical Bulletin, #TB288, Promega ( Chroma-Luc Series Reporter Vectors Technical Manual, #TM59, Promega ( Chroma-Glo Luciferase Assay System Technical Manual, #TM62, Promega ( EnduRen TM Live Cell Substrate.34mg E mg E , 34mg E6485 1,4, ViviRen TM Live Cell Substrate.37mg E , 3.7mg E , 37mg E6495 1,2, CellTiter-Glo Luminescent ml G7571 5, Cell Viability Assay ml G , ml G , Red Chroma-Luc TM Vector pcbr-basic Vector 2µg E , pcbr-control Vector 2µg E , 14