What is a FLIPR Assay?
FLIPR (Fluorescent Imaging Plate Reader) was developed to perform quantitative optical screening for cell-based kinetic assays. FLIPR incorporates an integrated design, including low-level optical detection, precise temperature control, and precise fluid handling, all in one package.
What is calcium flux assay?
The Calcium Flux assay can be used to monitor the activation of a GPCR via Gq second messenger in a live cell, non-imaging assay format. Calcium mobilization in PathHunter cell lines expressing Gq-coupled GPCRs is monitored using a calcium sensitive dye that is loaded into cells.
How does Fura 2 work?
This assay uses Fura 2-AM (Fura 2-acetoxymethyl ester, Invitrogen) to calculate intracellular Ca2+ within a single cell based on the relative emission between two excitation wavelengths (i.e., 340 and 380 nm), which automatically cancels out differences in dye loading and cell thickness.
What is Fura-2 imaging?
Fura-2 is a ratiometric and sensitive indicator dye for measuring intracellular calcium. Since its introduction in 1985, fura-2 has been cited in thousands of papers that describe its applications in a wide variety of cells.
What is ratiometric imaging?
Ratiometric fluorescence is the method where intensities at two or more wavelengths of an excitation or emission spectrum are measured to detect changes to local environment. Typically, a probe is used that is specifically sensitive to an environmental parameter such as ion concentration, pH, viscosity, or polarity.
What is flow cytometry assay?
Flow Cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument.
What is flow cytometry test for?
Flow cytometry can identify the type of cells in a blood or bone marrow sample, including the types of cancer cells. It detects types of cancer cells based on either the presence or the absence of certain protein markers (antigens) on a cell’s surface.
What is ratiometric fluorescence?
How does Fura-2 work?
Fura-2AM is a ratiometric dye. It can be excited by two wavelengths, 340 nm for calcium bound and 380 nm for unbound. Irrespective of the excitation wavelength, the dye will emit at 510 nm wavelength. The ratio of 340/380 is usually calculated for normalizing unequal loading of the dye into the cells.
What is a ratiometric indicator?
Ratiometric indicators are powerful tools for detecting and imaging fluxes in target ion concentrations. Calcium indicators, such as fura-2 and indo-1, are UV-excitable fluorescent molecules utilized for investigating the regulatory roles of calcium at a cellular level.
What is ratiometric sensing?
Ratiometric is used to describe an output signal which changes in proportion to a change input or supply voltage. A typical ratiometric device would be a strain gauge output pressure sensor, where the output sensitivity is described as a ratio between output and input supply voltage.
What buffer is used in FLIPR assay?
The standard assay buffer used in FLIPR™ experiments is HBSS with 20 mM HEPES supplemented with 0.5 mM Ca2+. The most common fluid addition volumes for a FLIPR assay are listed in Table 4.
What are the general guidelines for FLIPR™ assays for GPCRs?
Some general points regarding a FLIPR™ assay for GPCRs need to be noted: Some receptors contain trypsin-sensitive sites in their extracellular domain that results in a loss of response if the cells are harvested by trypsinization. In these instances, cells should be harvested by either scraping or using enzyme-free dissociation buffer.
What are the potential artifacts of the FLIPR test?
Potential Artifacts Although the FLIPR™ has facilitated advances in cellular calcium mobilization screens, these assays remain difficult to configure, relatively slow, and fraught with potential artifacts. Blocked FLIPR™ tips will lead to false positives in an inhibitor screen, or false negatives in an agonist screen.
What is FLIPR™ and how does it work?
The introduction of FLIPR™ (Fluorescence Imaging Plate Reader) in the 1990’s provided biologists with a fast and easy method of detecting G-protein coupled receptor (GPCR) activation through changes in intracellular calcium (Ca 2+) concentration.