ProcartaPlex Assays Support—Troubleshooting

Ideally, we recommend that you analyze the plates immediately. If the plate cannot be read on the day of the assay, it can be incubated overnight. Shake the plate for 30 mins at room temperature (600 rpm), then cover the plate and store on a level surface in the dark at 2-8 degrees C for reading the following day without significant loss of fluorescence intensity. When you are ready to read the plate, bring the plate to room temperature on an orbital plate shaker (600 rpm), still protected from light. Perform a wash step using 100 μL of fresh, room temperature Working Wash Solution/Reading Buffer and perform the analysis. We do not recommend storing the ProcartaPlex assay plate longer than 1 day.

All the components included in the ProcartaPlex kit are viable except the bead mixture. Storage of the beads at temperatures below 0 degrees C will damage the beads and render them unusable.

Waterproof markers that dry quickly will not affect the assay. However, markers that do not dry quickly might bleed into the wells during pipetting/washing which could influence the final readout.

Yes, it is possible to reread a finished ProcartaPlex plate without a loss in the signal or the number of beads counted. Please note that the Luminex instrument adds additional liquid to the wells with each analysis. It is possible that the wells may become overfilled with fluid after the third analysis. Hence, we do not recommend reading the ProcartaPlex plates more than two times.

Yes, our Universal Assay Buffer (Cat. No. EPX-11110-000) can be bought separately. We offer most of our ProcartaPlex buffers and reagents as stand-alone items. A complete list of what is currently available can be found on our website, under "Accessories".

Yes, you can use half a plate at a time, but make sure to seal the unused half with plate sealing tape to prevent any contamination during the assay. Alternatively, you can purchase extra plates (Cat. No. EPX-88182-000).

Yes, our Mix & Match team can adjust the bead regions as necessary to accommodate targets in your panel and accommodate instrument parameters (i.e., MagPix can only read 50 bead regions).

This will depend on your sample type. Often serum and plasma may require dilutions for certain targets whereas culture supernatant does not. However, it is optimal to combine only those targets that require the same dilution.

The TGF-beta1 assay requires an acid pre-treatment of the sample to reveal the TGF-beta1 protein and therefore, the assay cannot be combined with other assays. The acid pre-treatment of the sample will destroy the other protein epitopes. However the LAP TGF-beta1 assay does not require an acid treatment, but it will measure only the LAP-TGFbeta1 complex. Neither purified LAP nor purified TGF-beta1 alone can be measured in this particular assay.

Samples/beads could have been stuck in the flow cell. We recommend removing the 96-well plate and performing a wash and rinse cycle.

Here are possible causes and solutions:
- Samples and antigen standards not stored on ice. We recommend preparing the samples and standards on ice before setting up the assay.
- Contamination from re-using the Plate Seal. We recommend using a new Plate Seal for each incubation step.
- Incomplete washing. After adding the standards and samples, it is very important that any excess standards are removed during the wash step.
- Contamination from contents from adjacent wells. Avoid splashing the Wash Buffer during wash steps into adjacent wells.
- Poor pipetting techniques. We recommend using a multichannel pipettor and careful pipette techniques. Avoid touching pipette tips to sides of the wells when adding Wash Buffer.

The instrument may have been calibrated at high PMT settings. We recommend calibrating the instrument using the CAL2 Low RP1 target value.

Here are possible causes and solutions:
- Volume of the bead solution is too low. We recommend adding 120 µL Reading Buffer into each well and shaking at 600 rpm for 5 mins at room temperature to resuspend beads before reading on the Luminex instrument.
- High bead aggregation. We recommend vortexing the bead suspension well before using in the assay and ensuring that the beads are properly mixed during the incubation steps.
- Dyes contained in the beads are photo-bleached from overexposure to light. We recommend storing the bead solution and the 96-well plate in the dark.
- Samples causing the instrument to clog. We recommend removing the 96-well Flat Bottom Plate and performing a wash and rinse to the instrument and then re-running the assay with further dilution of samples.
- Probe height is incorrect. We recommend referring to the Luminex manual for proper adjustment of the needle height.
- The instrument needle is partially clogged. We recommend replacing or cleaning the needle according to the manufacturer's recommendations.
- Beads stuck to the bottom of the plate. We recommend confirming that the plate shaker is set to 600 rpm and shaking for at least 5 mins before reading.
- Air bubble in the sample loop. We recommend referring to the specific Luminex manual for your assay for proper removal of the air bubble.

Standards may not have been reconstituted and diluted correctly. We recommend preparing fresh antigen standards following the instructions provided in the corresponding ProcartaPlex user guide.

Here are possible causes and solutions:
- The wrong cell culture media may have been used to prepare the standards. We recommend using the same cell culture media that was used to culture the cells.
- The samples and antigen standards may not have been stored on ice. We recommend preparing the samples and standards on ice before setting up the assay.

This means that the data obtained in that run are below the detectable limit of the assay. Here are some possibilities and suggestions:

• Qualify your standard curve using the data analysis tools found in our Luminex Assays Support Center.
• Make sure that your dilution factors are set correctly.
• It is possible that the functional sensitivity of the standard curve can be extended to better distinguish the data at the lower end of the curve. This can be done by adding one or two further dilutions to the curve and re-running the assay with the samples of lower concentrations.
• It is possible that the levels of your protein of interest fall below the detection limits of the assay.