How To Automate MILLIPLEX^® Human Cytokine/Chemokine/Growth Factor Multiplex Panel A on the AAW™ Automated Assay Workstation

Intro To Automating MILLIPLEX^® Human Cytokine Multiplex Panel A Assay

MILLIPLEX^® Human Cytokine/Chemokine/Growth Factor Panel A enables simultaneous quantitation of up to 48 immune factors in human serum, plasma, cell culture supernatants, and other biofluids in a single well, in a 96-well plate format.

MILLIPLEX^® multiplex assays utilize a bead-based multiplex assay technology that can be automated on the modular AAW™ workstation powered by Opentrons^® with the help of BioTek^® plate washer technology and a Luminex^® detection system.

MILLIPLEX^® Human Panel A can be performed as either a 2-day protocol (overnight standard/sample incubation at 2-8 °C) or as a 1-day protocol (2-hour standard/sample incubation at room temperature).

Both approaches can be fully automated on the AAW™ Automation Assay Workstation, employing dedicated scripts for each protocol - Overnight Assay Day-1, Overnight Assay Day-2, and Same Day Assay. Automation streamlines serial dilution, plate lidding/un-lidding, on-deck shaking, and minimizes manual intervention. Off-deck plate washing (BioTek^® washer) and overnight incubation (a 2-8 °C plate shaker) are required hands-on steps, while the remaining assay operations are fully automated on the AAW™ workstation.

The following sections will detail the automation process from material preparation, protocol uploading, deck setup, and assay execution. It also provides verification and comparison of results between automated and manual workflows.

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Automation and MILLIPLEX^® Materials and Reagent Preparation

Instruments

Note: The AAW™ Automated Assay Workstation - Assay Ready platform automates both assay and sample preparation for a wide range of protocols. The system integrates robot hardware and modules to support automated MILLIPLEX^® assay workflows. The Assay Ready workstation comes with the 1- and 8-channel pipettes, gripper, heater-shaker, magnetic block, and deck expansion. It also grants access to the Millipore^® Protocol Library using standard Millipore^® and Sigma-Aldrich^® assay kits and reagents, ensuring consistent and reliable performance.

*Note: For the MILLIPLEX^® Human Cytokine Panel A + B Combo Pack, only Panel A can be automated with this protocol – Panel B will need to be run manually.

Preparation of MILLIPLEX^® Immunoassay Reagents

Amber tubes and black plate lids are used throughout to ensure protection from light exposure, replacing manual shielding methods such as foil or opaque lids. This sustains the stability of light-sensitive reagents (such as MILLIPLEX^® beads and Streptavidin-Phycoerythrin), supporting consistent assay performance.

Prior to assay setup, prepare kit reagents following MILLIPLEX^® Human Cytokine/Chemokine/Growth Factor Panel A kit manual:

1. Allow all reagents to reach room temperature.
2. Dilute 10X Wash Buffer to 1X using deionized water.
3. Prepare Antibody-Immobilized Beads (if supplied as individual vials).
4. Reconstitute lyophilized Quality Controls (QC1 and QC2) in 250 µL deionized water each.
5. Reconstitute lyophilized Standard with 250 µL deionized water.

Note: The robot will perform automatic serial dilutions of standards.

6. Reconstitute Serum Matrix in 1 mL deionized water, for experiments involving serum or plasma samples.

Available Analytes (48-Plex): sCD40L, EGF, Eotaxin, FGF-2, Flt-3L, Fractalkine, G-CSF, GM-CSF, GROα, IFNα2, IFNγ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-18, IL-22, IL-27, IP-10, MCP-1, MCP-3, M-CSF, MDC (CCL22), MIG, MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES, TGFα, TNFα, TNFβ, VEGF-A 

Serum and Plasma Sample Handling

Limit freeze/thaw cycles to two or fewer to preserve sample integrity. Refer to the MILLIPLEX^® kit protocol for sample preparation instructions.

Note: For running the RANTES analyte in multiplex, manually add the RANTES beads to the pre-mixed 47-plex beads (or 37-plex beads). RANTES beads are provided in a separate vial from the 47-plex (or 37-plex) mix. If testing for RANTES in Serum/Plasma, samples can be run singleplex and must be pre-diluted at 1:100 because neat Serum/Plasma contains a very high concentration of RANTES. RANTES should only be added to the mixed 47-plex (or 37-plex) beads if running samples other than Serum/Plasma.

Protocol Uploading and AAW™ Workstation Setup

Before uploading or running protocols on the AAW™ workstation, always review the user guide and all technical instruction to ensure safe and accurate operation.

Protocol Selection and Upload

Three verified kit-specific protocol scripts are available for MILLIPLEX^® Human Cytokine/Chemokine/Growth Factor Panel A from the Millipore^® Protocol Library:

1. Overnight Assay - Day-1 Protocol: Initiates assay setup and overnight incubation.
2. Overnight Assay - Day-2 Protocol: Completes processing detection incubation.
3. Same-Day Assay Protocol: Enables rapid, same-day completion without overnight steps.

Download the desired protocol from the Millipore^® Protocol Library and import the protocol file into Opentrons^® App on a computer. Transfer to the AAW™ workstation by selecting “Send to Opentrons Flex” or, if already loaded, use the touchscreen’s “Start Set-up” button for direct activation.

The workstation setup involves 5 primary screens displayed on both the computer and touchscreen.

• Parameters
• Hardware
• Labware
• Liquids
• Timeline

The first four screens guide instrument and assay configuration. The timeline screen provides detailed stepwise tracking and real-time progression during automated protocol execution.

Parameter Configurations

Protocol parameters are set on the AAW™ workstation to optimize the assay workflow and accommodate different sample types, plate configurations, and operational needs.

Complete the setting in the parameter screen and click “confirm values.” The typical parameters (varying by protocol) are listed below: 

*Note: It is recommended to run a dry protocol first to become familiar with the workstation steps and verify configuration before working with actual reagents. During a dry run, reagent mixing and incubation steps are shortened, and tips are returned to racks; this allows for safe, error-free protocol review and verification of setup.

Hardware Module Verification and Setup

The hardware module verification and setup process ensures that all pipettes, modules, and accessories are properly installed for automated MILLIPLEX^® protocols.

Install two liquid handling pipettes and the AAW™ Flex Gripper, and secure all deck hardware modules (trash bin, staging area, heater shaker) in their designated deck slots using the workstation touchscreen or app.

Confirm and install each hardware module in the specific deck position: 

After installing and securing modules, verify calibration and connection for each in the touchscreen/app. Resolve any errors or mismatches before proceeding.

Once hardware setup is confirmed and protocol parameters are set, continue to Labware and Reagent Loading as described in the next section.

Note: For MILLIPLEX^® protocols, the Opentrons Flex™ Heater-Shaker must be paired with the Universal Adapter “Type B”, installed into deck slot D1, and detected as active on the touchscreen/app. The protocol scripts preset the heater to room temperature (heater off) and set shaking speed to 700 rpm for on-deck incubation. By default, the protocol scripts will automatically open and close the shaker adapter latches to secure the assay plate or permit removal for washing and reading.

If manual intervention is required, the option to open or close the adapter Labware Latch is hidden by default and becomes visible in the touchscreen’s List View of labware (ASSAY PLATE/Opentrons Heater-Shaker Adapter, Slot D1).

Labware Preparation, Reagent Loading, and Deck Layout Setup

Labware Preparation

The labware setup feature allows selection and placement of all tubes, plates, reservoirs, tip racks, and modules on the robot deck, ensuring the system recognizes where each item is located for the protocol.

Identify and gather all labware required (see Materials section above) for the selected protocol, as specified in the Opentrons^® app or on the touchscreen. Specify and position all required tubes, plates, tip boxes, and reservoirs on the deck, with “Labware Position Check” to verify correct placement.

Note: Prior to starting a run, a Labware Position Check must be performed at least once by following the step-by-step instructions on the touchscreen or in the Opentrons^® app. Labware Position Check can be run with empty labware during a dry run or with reagent-ready labware. Once Labware Offsets are confirmed and applied to the selected protocol, the AAW™ workstation stores these values for future runs using the same protocol and Labware Position Check will not have to be repeated unless desired.

Reagent Loading

The Liquids setup displays the identity, volume, and source of each reagent or sample, as prompted by the touchscreen/app and protocol. Required reagent volumes are based on batch size and sample numbers entered in the parameters tab. The labware arrangement and reagent loading details for MILLIPLEX^® Human Panel A kit-specific protocols are shown in Figures 1-3.

Deck Layout Setup Configuration

With reagents prepped and loaded as described, the workflow now focuses on arranging each piece of labware into its designated deck slot for automated assay processing. Careful deck setup supports reagent conservation and protocol consistency, especially important when working with limited volumes or complex reagent handling. Before starting the protocol, verify tip rack placement, and confirm each location using the Labware Position Check, as shown in the touchscreen/app and reflected in Figures 4-6.

This configuration enables automated standard curve and sample dispensing for the overnight incubation workflow, reinforcing process reproducibility and minimizing manual intervention.

The black plate lid is included in the deck setup to provide essential light protection for the assay plate, replacing manual methods such as foil wrapping, aluminum sealing, or placing black lids on top of clear sealers. This automated step ensures consistent protection against light exposure during on-deck incubations with detection reagents, helping maintain assay integrity and reducing operator workload.

This protocol enables full walk-away automation for all incubation and detection reagent steps, while requiring temporary manual transfer of the assay plate to an off-deck washer for intermediate wash steps.

Deck layouts for all protocols (overnight and same-day) feature reagent-filled labware, assay plate, and tip racks, with automated lid placement for incubation as depicted in touchscreen and app displays. After confirming reagent and labware loading and hardware configuration, promptly begin the assay protocol with workflow continuity ensured by accurate deck position prompts. The automated AAW™ workflow for this MILLIPLEX^® Human Panel A assay significantly reduces manual handling and hands-on time, enabling streamlined, reproducible results.

Automated MILLIPLEX^® Human Panel A Assay Execution

After completing deck setup and labware loading, close the robot door and start the “run” by selecting the blue “Run” arrow. The AAW™ workstation will then execute the selected protocol.

Automated Assay Workflow Overview

The AAW™ workstation automates all critical initial liquid handling, standard curve preparation, sample transfer, reagent dispensing (matrix solution, Assay Buffer, Standards, QCs, samples, and Beads), and plate setup with robotic lidding, minimizing manual intervention for both overnight and same-day protocols (~20 min, dependent on sample count).

• For the overnight protocol, after the robotic setup and manual plate sealing, the user walks away during the off-deck overnight shaking incubation at 2-8 °C.
• For the same-day protocol, the robot completes all setup, reagent transfers, and on-deck lidding, followed by a continuous walk-away period of > 2 hr 20 min until manual plate wash is needed.

After incubation, plate washing is performed off-deck (BioTek^® washer). The AAW™ workstation then resumes automated, hands-off reagent additions (Detection Antibodies and SAPE), with robotic lidding/de-lidding ensuring > 1.5 hours of uninterrupted automation. Final steps include automated xMAP^® Sheath Fluid PLUS addition (with lidding and on-deck shaking) and data acquisition on a Luminex^® instrument.

This automated workflow maximizes hands-off operation and walk-away convenience at every feasible step, supporting high-throughput performance, reproducibility, and scalability. The overall stepwise protocol comparison is visually summarized in Figure 7.

The next section presents the verification data and comparative results obtained from these manual and automated assay runs.

Results and Verification: Automated MILLIPLEX^® Assay Vs Manual Assay

Automated MILLIPLEX^® Human Cytokine Panel A assays run on the AAW^™ workstation were directly compared with established manual protocols to verify assay performance, precision, and sample result equivalence. All QC and test results were evaluated against manufacturer specifications to confirm that the automated workflow meets required precision and performance standards.

Quality Controls and Intra-Assay Precision

Quality Control Pass Rates

Each automated assay run included two kit-provided quality controls (QC1 and QC2), assessed against the manufacturer’s lot-specific acceptance ranges. For all 48 analytes, both QC1 and QC2 results obtained from the automated platform consistently fell within the specified kit’s QC acceptance ranges, confirming reliable performance and robust integration of the automated protocol.

Intra-Assay Precision

Precision was evaluated by analyzing eight replicates of QCs (n=8, in duplicate wells) for all analytes, using both manual and automated AAW™ protocols. The automated assay consistently achieved intra-assay precision, as indicated by percent coefficient of variation (%CV) below 15% for all analytes and below 10% for most, meeting the accepted benchmarks and paralleling manual protocol performance. These results demonstrate that the automated assay is precise and reproducible, supporting scalable and reliable laboratory workflows.

Sample Correlation: Automated Protocol vs Manual Protocol

Thirty-eight human serum samples were analyzed side-by-side using both manual and automated protocols in the overnight format, with the 47-plex panel (RANTES omitted due to special dilution requirements). The coefficient of determination (R²) was used to assess agreement between automated and manual protocols, with R² > 0.95 as the acceptance criterion.

Twenty-four analytes in the serum sample cohort exhibited biomarker levels spanning a broad dynamic range, enabling robust correlation analysis between automated and manual protocols; strong sample correlation was demonstrated for these analytes through scatter plots of manual (x-axis) versus automated (y-axis) assay results.

Figure 8 presents representative analytes (n=6) exhibiting regression slopes between 0.80 and 1.20, with R² > 0.95, consistent with field standards for acceptable method correlation and proportionality and confirming matched quantitative performance between automated and manual runs. Additionally, the automated method can process multiple samples with reduced risk of human error, resulting in greater reliability and efficiency for high-throughput applications.

Summary

The AAW™ workstation is a robotic liquid handler with a user-friendly interface, designated to automate high-throughput MILLIPLEX^® multiplex immunoassay workflows such as MILLIPLEX^® Human Cytokine Panel A, and significantly reduce hands-on time. The system delivers verified assay precision and consistency, with QC pass rates and intra-assay variability matching or exceeding manual protocols. By seamlessly automating manual assay procedures, including sample loading, reagent dispensing, and incubation, the AAW™ workstation offers a robust platform for multiplex cytokine quantitation and routine biomarker studies.

Integrated with an automatic washer module, the AAW™ workflow further enhances sample throughput and standardization by automating all assay steps except for the off-deck wash and overnight incubation steps. This robust approach yields reproducible results with low %CV and sample detection near or surpassing manual protocols, making it an ideal solution for scalable biomarker analysis in various research fields.

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Tips & Tricks

• Before starting, turn off AAW™ workstation LED lights and the heater function on the heater-shaker (heater off is pre-programmed in the MILLIPLEX^® protocol scripts) to prevent unwanted light exposure and temperature changes during assay setup.
• Run a dry practice protocol (with water or buffer only) to become familiar with instrument operation; dry runs also help optimize (shorten) mixing and incubation steps and support efficient tip return to rack.
• If dispense tips are off-center in wells, use Labware Position Check to adjust and ensure correct location before actual assay run.
• (Optional) Use an aluminum plate seal for the assay plate during Day 2 detection and SAPE incubation if no dedicated plate lid is available, and fold tabs to cover plate edges for full protection.