Signosis Innovative Plate Assay Solutions Illuminated Pathways (For Research Use Only) Catalog #: BA-1001 (Hela), BA-1002 (HEK293) BA-1003 (HepG2) Introduction Cellular signal pathways can be activated through treating cells with different stimuli. To discover differences in signal pathways, to delineate molecular players in complicated pathways, or to elucidate proteins that regulate the pathways, one can compare cells of different genetic make-up, such as normal versus cancer tissues, wild-type versus gene knockout within a specific pathway, or wild-type versus mutation of a specific gene. Signosis now provides the Illuminated Pathways - This kit contains multiple pairings of a stimulus reagent and its corresponding luciferase reporter—one for lighting a specific pathway and another for monitoring the activation. The individual pre-packed TF reporter reagents are pre-coated in a 8X12 arrangement on a sterile 96-well white plate. You just need to add your cells and treat with the inducers provided. The white plate is ready for luciferase reading in a luminometer. Materials provided Two pre-coated sterile 96-well white plates 6 inducers Materials required but not provided (May be substituted with a comparable third-party product) Luciferase substrate -- Signosis P/N LUC100 Passive lysis buffer -- Promega P/N E-1941 Assay procedure NOTE: Before removing the foil in the tissue culture hood, centrifuge the 96-well plate at 1,000 RPM for 30 seconds in a swinging-bucket rotor. Make sure to balance the plate with a similar counterweight or the plate and centrifuge may sustain damage. Principle This kit utilizes 8 recombinant viruses that are generated from insect cells with a modified baculovirus system that contains mammalian delivery element, TF consensus sequence and luciferase reporter gene. The recombinant viruses are pre-coated in 96-well sterile white plate to simplify the assay. Experimentally, seed the cells overnight in the 96-well plate at 50%-70% confluency. Then treat with the inducers, followed by luciferase assay. This array can be used in routine lab settings without additional or special safety protocols as these recombinant viruses do not infect or replicate and are easily inactivated with 70% ethanol in case of a spill. 1. Handling upon arrival 3. It is strongly recommended that you store Illuminated Pathway plates at 4°C in the dark as soon as possible upon arrival. Do not store in -20°C. Store the inducers in -80°C. IMPORTANT: Baculovirus are light-sensitive and require gentle handling. Avoid vortexing and frequent pipetting as mechanical forces can damage the virus and decrease the viral titer. The day before performing the assay, trypsinize the cells and seed 100uL per well of the precoated 96-well sterile white-wall plate at 50-70% confluency. Note: 5,000 to 10,000 cells per 100uL yields approximately 50-70% confluency for most cultured cell lines. Please empirically determine the cell density for your cells of interest before each experiment. 2. 4. 5. 6. 7. 8. Incubate the plate in a humidified incubator at 37oC with 5% CO2 overnight to 24 hours. No media change necessary. Add appropriate treatment to cells to induce luciferase activity. Remove the media by aspiration and add 100l of PBS to each well. Remove PBS by aspiration and add 20l of passive lysis buffer to each well. Incubate cells in lysis buffer for 15 minutes at room temperature. Add 100l of luciferase substrate to each well and gently pipette up and down. Immediately read the plate in a luminometer. NOTE: Shelf life is 3 months. Use all wells at the same time and do not save some for a later time. Signosis, Inc. • 1700 Wyatt Drive Suite 10-12 • Santa Clara, CA 95054 • Tel 408 747 0771 • Fax 408 864 2182 • [email protected] Plate Diagram for Illuminated Pathways for Hela (BA-1001) A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 NFkB NFAT TCF/LEF GAS-ISRE AP1 p53 Negative Positive NFkB NFAT TCF/LEF GAS-ISRE AP1 p53 Negative Positive NFkB NFAT TCF/LEF GAS-ISRE AP1 p53 Negative Positive NFkB NFAT TCF/LEF GAS-ISRE AP1 p53 Negative Positive NFkB NFAT TCF/LEF GAS-ISRE AP1 p53 Negative Positive NFkB NFAT TCF/LEF GAS-ISRE AP1 p53 Negative Positive NFkB NFAT TCF/LEF GAS-ISRE AP1 p53 Negative Positive NFkB NFAT TCF/LEF GAS-ISRE AP1 p53 Negative Positive NFkB NFAT TCF/LEF GAS-ISRE AP1 p53 Negative Positive NFkB NFAT TCF/LEF GAS-ISRE AP1 p53 Negative Positive NFkB NFAT TCF/LEF GAS-ISRE AP1 p53 Negative Positive NFkB NFAT TCF/LEF GAS-ISRE AP1 p53 Negative Positive 8 9 10 11 12 Plate Diagram for Illuminated Pathways for HEK293 (BA-1002) 1 A B C D E F G H 2 3 4 5 6 7 NFkB NFkB NFkB NFkB NFkB NFkB NFkB NFkB NFkB NFkB NFkB NFkB NFAT NFAT NFAT NFAT NFAT NFAT NFAT NFAT NFAT NFAT NFAT NFAT TCF/LEF TCF/LEF TCF/LEF TCF/LEF TCF/LEF TCF/LEF TCF/LEF TCF/LEF TCF/LEF TCF/LEF TCF/LEF TCF/LEF CREB CREB CREB CREB CREB CREB CREB CREB CREB CREB CREB CREB AP1 AP1 AP1 AP1 AP1 AP1 AP1 AP1 AP1 AP1 AP1 AP1 HIF HIF HIF HIF HIF HIF HIF HIF HIF HIF HIF HIF Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Plate Diagram for Illuminated Pathways for HepG2 (BA-1003) 1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12 NFkB NFkB NFkB NFkB NFkB NFkB NFkB NFkB NFkB NFkB NFkB NFkB NFAT NFAT NFAT NFAT NFAT NFAT NFAT NFAT NFAT NFAT NFAT NFAT SMAD SMAD SMAD SMAD SMAD SMAD SMAD SMAD SMAD SMAD SMAD SMAD GAS-ISRE GAS-ISRE GAS-ISRE GAS-ISRE GAS-ISRE GAS-ISRE GAS-ISRE GAS-ISRE GAS-ISRE GAS-ISRE GAS-ISRE GAS-ISRE AP1 AP1 AP1 AP1 AP1 AP1 AP1 AP1 AP1 AP1 AP1 AP1 HIF HIF HIF HIF HIF HIF HIF HIF HIF HIF HIF HIF Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Signosis, Inc. • 1700 Wyatt Drive Suite 10-12 • Santa Clara, CA 95054 • Tel 408 747 0771 • Fax 408 864 2182 • [email protected]
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