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Our multiplex immunoassays for flow cytometers allow simultaneous measurement of many proteins at the same time in each well. At the heart of these assays is the innovative FirePlex particle technology that provides this multiplexing ability.
Our FirePlex particles have 75 different codes, significantly more than it is possible to distinguish with conventional bead-based immunoassays for flow cytometers.
For both conventional bead and FirePlex particle multiplex immunoassays, each analyte in a panel must use a particle with a different code so your analysis software can correctly assign each signal to the right analyte. More codes means you are more likely to be able to analyze all of your proteins in one panel.
Particles are made from a porous bio-inert hydrogel that allows target capture throughout the 3D volume. Compared with the leading bead-based platform, FirePlex particle technology provides >100x more binding sites per particle.
More binding sites means assays with a broader dynamic range, so more analytes can be quantified in the same well, with no need for multiple sample dilutions.
Particles contain three distinct functional regions each separated from the other by inert spacer regions. The central analyte quantification region contains probes that capture target proteins. The end regions function as two halves of a barcode to distinguish different particles.
End regions encode particles
Central analyte quantification region
Particle structure. Distinct regions capture target analytes and encode particles.
When particles are passed through a flow cytometer, they appear as a series of three cells. By separating the target quantification region from the particle encoding information, we reduce cross-talk between channels and eliminate the need for compensation.
The flow cytometer and FirePlex Analysis Workbench use information from the three regions to generate abundance data for each analyte:
Particle presence is triggered in the green channel. Events from all three regions are detected in the green channel to signify the presence of a particle.
Fluorescence intensity in the central region determines protein quantity. The flow cytometer detects a fluorescent signal in the red channel from the center of the particle. This comes from captured and labeled target analytes and is proportional to the amount of the analyte present in the sample.
A fluorescent code to distinguish different analytes. By containing two end regions of varying fluorescence intensity, a particle code is generated in the yellow and green channels. Up to 70 codes are possible, and in every panel each analyte is assigned one of those codes so that data from a mixture of particles can be attributed to individual target analytes.
Software to stitch all the data together. After acquisition, the FirePlex Analysis Workbench software takes events from the three regions of the particles and stitches them back together into a single event, giving you simply the abundance data for each target analyte.
NB the FirePlex technology was previously known as the Firefly multiplex immunoassay / Firefly multiplex miRNA assay technology.
FirePlex® is a registered trade mark in the United States and is an unregistered trademark elsewhere.