Cells, Membranes and Assay Kits

highly characterized reagents for screening and profiling

in multiple physiologically relevant assay formats

Stable cell lines

  • The best tools for studying all GPCR signaling-biases in one cell line
  • Minimally modified GPCRs and other targets directly selected in functional assays
  • Validated in MultiScreen™ assay kits in 384-well format
  • Holding the industry’s highest quality standards
  • Rigorous quality control ensures that 99% of the stable cell lines pass 2-month stability

Division-arrested cells

  • Convenient tools for cell-based high-throughput screening
  • Plate and read with ready-to-go assay conditions
  • Low-cost evaluation of a stable cell line or for limited number of compounds
  • Derived from well-characterized stable cell lines
  • Quality control performed in each lot for high performance functionality

Membranes preparations

  • Ideal for receptor binding assays
  • High receptor density
  • Prepared from Multispan’s stable cell lines for minimal lot-to-lot variability
  • Two-week turnaround for rapid delivery
  • Quality control by FACS for receptor surface expression

Summary Catalog

MULTISCREEN™ TR-FRET cAMP 1.0 No-Wash Assay Kit

INTRODUCTION

Multiscreen™ TR-FRET cAMP 1.0 No Wash Assay Kits provide a homogenous TR-FRET assay method for adenylyl cyclase activity detection. α-cAMP antibody (Ab) is labeled with MultiScreen™Eu while cAMP is labeled with MultiScreen™650. In the absence of cAMP, MultiScreen™650-cAMP is bound to MultiScreen™Eu-α-cAMP-Ab to give a strong TR-FRET Emission at 655 nm. Free cAMP in the test sample competes for binding to the MultiScreen™ Eu-α-cAMP-Ab, reducing TR-FRET signal from MultiScreen™650-cAMP binding. The MultiScreen™650 labeled cAMP only has fluorescence lifetime of nanosecond while MultiScreen™Eu-labeled α-cAMP antibody has much longer fluorescence lifetime value due to the TR-FRET. The magnitude of TR-FRET is proportional to the concentration of cAMP in a sample. The assay can be performed in 96- or 384-well microtiter-plate format and adapted to automation.

Protocol Img 1

PREPARE WORKING SOLUTIONS (Mix well by gentle-vortexing or pipette mixing after each step)

1. cAMP standard: Add 100 μL Component E to Component C to make 1mM stock solution for MSCM01-10 and MSCM01-100 kits only and gently vortex to mix: Add 1 μL 1mM stock solution into 99 μL Component E or cell culture media to make 10μM standard followed 4-fold serial dilution to make 10000, 2500, 625, 156.3, 39.1, 9.8, 2.4, 0.61, 0.15, 0.038 nM final concentrations. Mix gently with a pipette after each dilution. Add 10μL or 20μL of serial diluted cAMP standard per 384-well in microtiter assay plate after last the incubation period from step 5.

2. MultiScreen™Eu-α-cAMP-Ab working solution: Add 50μL Component A to 2.5mL Component D (scale down based on need). Prepare right before use. Store at 4°C.

3. MultiScreen™650-cAMP working solution: Add 50μL Component B to 2.5mL Component D (scale down based on need). Prepare right before use. Store at 4°C.

cAMP ASSAY PROTOCOL (384-well format)

4. Prepare cells (Evaluate each cell line to determine optimal cell density and other conditions.)

Adherent: Plate cells overnight in growth media with 10% FBS at 3k-9k cells/40 μL/384-well in Poly-D-Lysine coated white opaque bottom plate. Remove growth media carefully before compound treatment.

Suspension: Centrifuge the cells from the culture media and then suspend the cell pellet in the appropriate amount of Assay Buffer at 3k-12k cells/5 μL/ 384-well in small volume, white, opaque bottom plate.

5. Compound Treatment (The incubation time and temperature can be optimized for each receptor): Add 15μL of test compounds to adherent cells or 5μL to suspension cells per well and incubate for 20 minutes at 37°C.

6. Termination (30μL or 20μL final volume): Add 7.5μL MultiScreen™650-cAMP working solution and 7.5μL MultiScreen™Eu-α- cAMP-Ab working solution sequentially to adherent cells per well or 5μL of each on sequentially to suspension cells per well. Incubate 30 minutes at room temperature in the dark. Read fluorescence emission on a TR-FRET compatible reader at 665 nm and 620 nm.

Protocol Img 2