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FlowCytoMix technology uses fluorescent latex beads coated with capture antibody to quantify multiple proteins simultaneously (20) in a single tube. The methodology significantly reduces sample volumes of analytes to 25ml and time compared to the traditional ELISA and Western blot techniques. The technology has the advantage over other immunobead based assays in that you just need a single argon 488nm laser with 3 detectors. The analysis software is easy to use and freely downloadable from the Internet, see link.
How FlowCytomix™ Works
Kits contain fluorescent bead sets, each pre-coated with a unique antibody specificity. Within two bead size populations, A and B (4 μm & 5 μm), there are multiple bead subsets, differentiated by varying intensities of an internal fluorescent dye. The dye can be excited by an Argon, He-Ne, or UV laser, and emits at 690 nm (the far red spectrum), which is detected in the FL-3/FL-4 channel. The combination of the two different bead sizes and different internal dye intensities makes it possible to distinguish up to 20 bead sets in one fluorescent channel. Streptavidin-PE, which binds to the biotin conjugate, emits at 578nm and is detected in the FL-2 channel, allowing the quantification of the analyte.
FlowCytoMix assays
These kits contain 4 & 5um latex beads coated with specific antibodies (up to 20 different ones at anyone time). Each of the antibody capture beads have a variable amounts of group differentiation dye which emits at 690nm after excitation at 488nm. These capture a wide range of cytokines and soluble factors in serum, plasma and cell culture supernatants. After the initial incubation a biotin conjugated antibody is incubated with the beads optimally paired to specifically bead bound analytes. The reporter fluorochrome PE bound to strepavidin is then incubated with beads. The samples are then ready to run on most flow cytometers, see pdf.
Standard Curves
Each FlowCytoMix kit comes with 7 standards for each analyte within a kit. The Human Th1/Th2/Th17 kit comes with antibody capture beads for IL-6, 8, 10, 2, 17A, IFNg, IL-12p70, TNFb, TNFa, IL-1b, IL-5 and IL-4, see figures.
