Optimization of Bead Loading for MAGPIX®
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Optimization of Bead Loading for MAGPIX® MAGPIX® Chamber Capacity Luminex Flow Cytometry vs. MAGPIX® Figure 1: Flow Cytometry vs. Flourescent Imaging Flow Cytometry-Based Analysis Interrogate label with green laser (525 nm) LED/Image-Based Analysis Interrogate bead with red LED (635 nm) Identify and quantify with CCD imager Interrogate label with green LED (525 nm) Magnetic Capture Figure 2: MAGPIX® Chamber Loading Capacity Chamber Capacity Linear Range Total Classified Events At lower bead concentrations, the Total Classified Events increase linearly with the number of beads in the Sample Load Volume. As the bead concentration loaded into the chamber increases, the image processing algorithms start to throw away beads that are interfering with each other, and the Total Classified Events do not increase as fast as the concentration. As the bead concentration approaches the chamber capacity, the Total Classified Events stop increasing with increased bead concentration. If the chamber is overloaded, the Total Classified Events will decrease. Identify bead region based on internal dye concentrations Sheath Fluid MAGPIX Bead Loading The MAGPIX chamber has an upper end capacity of ~60,000 65,000 beads. Since MAGPIX loads all the beads in the Sample Load Volume into the chamber, the total number of beads in the Sample Load Volume is limited by this threshold. As a general rule, the end user should target a final count of 1,250 beads per region in the Sample Load Volume. (See sample load examples on page 3.) Quantify binding events Interrogate bead with red laser (635 nm) In a Luminex flow cytometer, beads are injected single file through a cuvette and are counted and classified as they pass by a laser. When the bead count for all regions reaches the desired value, the system stops acquisition and moves on to the next well. In the MAGPIX® system, the entire Sample Load Volume is injected into a chamber where the beads are imaged. The image is then processed by the software, and the data is presented to the user. Chamber Loading Range Chamber Overload Classification Capacity Total number of beads loaded in the chamber (Final Bead concentration x Sample volume uptake) Multiplex equivalent (assuming a target load of 1,250 beads for all region

The MAGPIX system pushes the entire Sample Load Volume into the chamber. Thus, the images can have too many beads in them to give accurate results. The image-processing algorithms favor the brighter beads over the dimmer beads. In higher plex assays, the lower intensity regions will have fewer counts than the high intensity regions. As the chamber becomes overloaded, the user will see the DotPlot start to drop in the lowest CL1 and CL2 regions first. Figure 3-A shows a 50-plex DotPlot with 1,250 beads per region in the Sample Load Volume; Figure 3-B shows the same DotPlot with 3,750 beads...

Figure 4: Bead Count Optimization Volume of bead mix used per well Initial bead concentration (per region) Controlled by assay protocol Number of beads added to the well (per region) Controlled by operator and wash method Final resuspension volume Bead retention efficiency Sample volume Final Bead concentration (per region) Target load (per region) Number of beads loaded in the chamber 1,250 beads (per region) Target count (per region) Guarantee at least 50 bead count per region Successful assay

Figure 4: Bead Count Optimization Sample Volume Number of Beads Loaded (Per Region) Total Number of Beads Loaded (For a 50-Plex) Sample Volume Number of Beads Loaded (Per Region) Total Number of Beads Loaded (For a 50-Plex)

If bead concentration is uncertain: 1. Create protocol and batch using an estimate concentration and the guidance table to define “Sample Volume”: Example: • The Assay protocol does not specify the initial bead concentration, so it is estimated at 2,000 beads per region per 100 μL. • The final resuspension volume is specified by the protocol at 150 μL. • The guidance table suggests a “Sample Volume” between 75 μL to 100 μL to reach the target load of 1,250 beads per region. Assuming there maybe some bead loss during the assay preparation process, the sample volume of 100 μL is chosen. 2....

6. Specify a new Sample Load Volume (allowed range 20 to 200 μL). The change is logged into the “Current Batch” display. Caution: The change is implemented for the current batch only. The protocol used to create the batch remains unchanged. 7. Resume the batch. 8. After next well, the system pauses automatically again. 9. If needed, repeat steps 5-8 until bead loading is optimized. 10. Once the bead counts are optimized, and the DotPlot is satisfactory, unselect the “Single Step” feature and resume the acquisition of all remaining wells without automatic pause implemented. For more...