HomeMILLIPLEX® Multiplex for Luminex® ImmunoassaysTips & Tricks: Equipment Settings and Data Analysis for Running MILLIPLEX® Multiplex Assays

Tips & Tricks: Equipment Settings and Data Analysis for Running MILLIPLEX® Multiplex Assays

Need some advice on choosing equipment settings or performing data analysis when running MILLIPLEX® multiplex assays? Check out these tips and tricks from the experts, including Luminex® multiplex instrument settings, general instrument considerations, and data analysis tips below.

General Equipment Settings for Luminex® Multiplex Instruments

The following describes tips on equipment settings for Luminex® multiplex platforms including the xMAP® INTELLIFLEX, Luminex® 200™, FLEXMAP 3D®, and MAGPIX® instruments.

  • For the MAGPIX® system, choose the “enhanced startup” setting instead of the common startup.
    • This will ensure proper calibration and cleaning prior to running the assay.
    • Working with serum can be “stickier” than other biological fluids and can affect the performance of the instrument unless it is properly cleaned.
    • Luminex® experts can provide a recommended protocol for maintenance.
  • Be sure the needle probe is clean. This may be achieved by sonication and/or alcohol flushes.
  • Probe height:
    • xMAP® INTELLIFLEX System: Adjust the probe height based on the type of plate you are using and place an alignment disk or an alignment sphere in the well according to the protocol recommended by Luminex® experts.
    • Luminex® 200™ System: Adjust the probe height according to the protocols recommended by Luminex® experts to the kit solid plate using 3 alignment discs.
    • FLEXMAP 3D® System: Use the probe height adjustment tool (white plate) that has spots for both the 384-well and 96-well plates.
      — The 96-well kit plate well dimensions (3.5 mm) require using location F12 on the white probe height adjustment tool.
    • MAGPIX® System: Adjust the probe height according to the protocols recommended by Luminex® experts to the kit solid plate using 2 alignment discs.
  • Annotating control wells on the instrument can be tedious, with a lot of manual typing.
    • It is possible to enter the wells as unknowns instead of controls to avoid typing in the annotations for controls, then comparing with your chart later.
  • It is important to use the manufacturer’s specific gate settings.
  • The Luminex® 200™ system’s xPONENT® acquisition software has two functions: one for magnetic (MagPlex®) beads and one for nonmagnetic (MicroPlex®) beads.
    • Be sure to select MagPlex® beads to run MILLIPLEX® multiplex kits.
    • If the wrong type is selected, the plate does not need to be reread. The batch can be replayed with the corrected protocol setting.
  • For information on xPONENT® software and to request software templates contact Technical Support or your Sales Specialist.
  • If a plate cannot be run immediately (within 4 hours) (e.g., it needs to be taken to another site to run the assay), suspend your sample in Sheath Fluid PLUS or Drive Fluid PLUS or assay buffer.
  • Ten plates can be run with one bottle of Drive Fluid PLUS for the MAGPIX® system.
  • To change a standard curve, for example, from a 7-point curve to an 8-point curve, simply make a new protocol and replay the batch.

Running MILLIPLEX® Kits on Other Luminex® Instruments

  • Luminex® 200™, FLEXMAP 3D®, and MAGPIX® systems with xPONENT® software and xMAP® INTELLIFLEX systems with the INTELLIFLEX Software are required to run a MILLIPLEX® multiplex assay.
    • If you want to try a kit before purchasing an instrument, ask your Sales Specialist to provide a demonstration using the Luminex® platforms and MILLIPLEX® kits.
    • Magnetic bead assays cannot be run on any instruments using Luminex® IS 2.3 or Luminex® 1.7 software.
  • Since all Luminex® machines (xMAP® INTELLIFLEX, Luminex® 200™, FLEXMAP 3D®, and MAGPIX® instruments) are built by Luminex® Corporation, MILLIPLEX® kits can be run on any of these machines, regardless of the name given to the machine by a Luminex® business partner.
  • If using Luminex® instruments with software other than xPONENT® software (Bio-Plex® Manager™, MasterPlex®, STarStation, LiquiChip, LABScan™ 100 software), follow instrument instructions for gate settings and additional specifications from the software vendors for reading assays using Luminex® magnetic beads.
  • To read a MILLIPLEX® kit on a Bio-Plex® instrument, select 5K-25K for magnetic beads, depending on the version of Bio-Plex® Manager™ software.

Instrument Considerations for Running MILLIPLEX® Assays

Starting up and shutting down your system correctly will ensure its longevity. The instructions for the xMAP® INTELLIFLEX, Luminex® 200™, and MAPGIX® systems are located within the systems’ user manuals:

  • xMAP® INTELLIFLEX User Manual, page 40
  • Luminex® 200™ User Manual, Section 3, page 17
  • FLEXMAP 3D® User Manuel, page 30
  • MAGPIX® User Quick Guide 4.2.

Short-term cleaning will prevent sample-induced clogging, while long-term cleaning is important to ensure that Sheath Fluid PLUS or Drive Fluid PLUS does not evaporate and crystallize.


  • Check probe and insert into the reader. Set probe height.
  • Fill reservoirs (Milli-Q® water, 70% EtOH, 0.1M NaOH).
  • Revive instrument: revive from storage, daily start-up.
  • Calibrate and verify instrument: system installation.
  • Read the entire kit protocol.
  • Acquire “Materials Required But Not Supplied”.
  • Confirm accuracy of pipettes.


  • Follow the kit protocol.
  • Set up experimental design on acquisition software.
  • Run assay.
  • Run “Post Batch Routine”.


  • Daily shutdown (overnight):
    • Run “Clean Routine”.
    • Run “Clean Routine”.
    • Run “Daily Shutdown Routine”.
    • Remove probe and clean in a sonicating water bath.
  • Long-term shutdown (longer than one week):
    • Run “Clean Routine” multiple times.
    • Run “Prepare for Storage” part 1.
    • Prime multiple times with Milli-Q® water (use an empty sheath fluid container).
    • Run “Prepare for Storage” part 2.
    • Remove probe and clean in a sonicating water bath.

Data Analysis Tips

Bead Counts

We recommend counting 50 beads when running MILLIPLEX® assays.

  • According to Luminex® experts, a minimum of 35 beads per region need to be counted.
  • Fewer than 35 beads could cause a shift in the MFI (Median Fluorescence Intensity) value of the bead population.
  • MFI will not change for bead counts ≥35.
  • Don’t worry if there is a 35-bead count on one bead region and 400 for others. MFIs will not be affected.

Percent Coefficient of Variation (%CV)

  • High %CVs for standards or samples can be due to low bead count.
  • Our target intra-assay %CV is <15% and our target inter-assay %CV is < 20%.

Calculating Lower Limit of Quantification (LLOQ) and Upper Limit of Quantification (ULOQ)

  • Defining LLOQ and ULOQ requires a tight standard curve (e.g., a 1:2 or 1:3 serial dilution to the point that you achieve saturation at both ends).
  • Choose the lowest and highest standard curve points that have a recovery of +/- 20%.
  • Verify that this is the LLOQ and ULOQ by running 5 assays with the LLOQ and ULOQ as samples against a curve using the assay serial dilution factor where the lowest standard is below LLOQ and the highest standard is above ULOQ.
  • Inter-assay precision should be within 20% for LLOQ and ULOQ samples.

Curve Performance/Fit

  • Standard point %CVs should be <15%.
    • High %CVs here indicate improper technique was used when making standard curve dilutions. Examples of poor technique include:
      • Not vortexing between tubes.
      • Not vortexing while loading the plate.
      • Not pipetting equal amounts into the plate.
    • The lower the concentrations of analytes, the higher the %CVs tend to be. With new users, this improves with time and practice.
    • For any standard points that have high %CVs, samples in that range of the curve should be interpreted with caution.
    • Alternatively, a standard point or one of the replicate wells can be flagged/masked, although it can be difficult to decide which well to flag if only duplicates are run.


  • Percent recovery should be 100% +/- 30% (industry standard), although some researchers will have their own acceptance criteria.
  • Percent recovery is usually worse at either extreme of the curve, but this also improves with time and practice.
    • For curve statistics, focus on the R2 value, which approaches, but never equals unity. (Note that an R2 value of “1” is seen with software rounding of 0.9999).

Minimum/Maximum Detectable Concentration (minDC/maxDC)

  • For many assays, the minDC/maxDC will be outside the standard points (extrapolated) due to good curve performance and fit.
  • To avoid seeing extrapolated data, set the desired range of detection in data analysis software.
    • Deciding whether to use the “Best Fit” vs. 5-parameter lot option depends on your comfort level to determine how appropriate it is to “play” with curve fit to find the best one.
    • If samples fall above the dynamic range of the assay, dilute the samples further with the appropriate matrices/media and repeat the assay.

How To Monitor/Avoid Lot-to-Lot Drift

  • MILLIPLEX® standard points maintain consistent values from lot to lot.
  • Lot-to-lot drift is monitored and mitigated using full-curve comparison and comparing the relative potency of each analyte against a reference lot.
  • All data are compiled in a single database, and trend charts are maintained in our records.

Easy Multiplex Data Analysis with Belysa® Immunoassay Curve Fitting Software

We offer a powerful combination for analyzing multiplex immunoassay data, coupling our Belysa® Immunoassay Curve Fitting software with data acquired using the Luminex® xPONENT® software. Belysa® software enables you to manage, track, and analyze your multiplex assays rapidly and efficiently, giving you more time to focus on advancing your research. Data acquisition and analysis integrate seamlessly with all Luminex® xMAP® instruments.

  • Step 1: Drag and drop your .csv file obtained from the xPONENT® acquisition software.
    • Belysa® software will accept acquisition files from all RUO Luminex® instruments. Files can be dragged into the open browser to be opened in the Belysa® platform.
  • Step 2: Examine and automatically optimize the curve fit for your data.
    • Belysa® tools will parse out the raw data and present it to the user, annotating the curve with Standard, Control, and Sample points. Through a simple wizard function, the software will provide the best fit for the acquired data. A manual curve fit is also an available option.
  • Step 3: Scan data to ensure replicate hygiene.
    • Belysa® software notifies the user in two ways:
      • Belysa® tools alert the user to data that is at the low end of the assay range (e.g., below the limit of quantitation or non-detectable).
      • Belysa® tools also flag technical errors within an assay, whether at the raw data level (such as bead count) or in calculated parameters (such as recovery or %CV).
  • Step 4: Compare standard curves against a previous experiment to confirm mathematical similarity.
    • Comparing standard curves’ slopes between batches or runs ensures that different kits perform equally, either within a run or through the course of a longitudinal study. The user first selects a curve to establish as a reference against which subsequent assay curves are compared. If the calculated slope ratio is equal to 1, then the curves are statistically similar.
  • Step 5: Export your data.
    • Each experimental mode in the software offers a slightly different report structure, single analyte, and multi-analyte. These are available in .csv, .txt, Excel, and PDF for future analysis or record keeping.

Learn more about Belysa® software in our Monitoring Immunoassay Method Reproducibility article.

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