COMP Human ProcartaPlex™ Simplex Kit - FAQs

Unfortunately, we do not currently offer a Mac-compatible version of the ProcartaPlex Analyst Software. ProcartaPlex Analyst Software is compatible with PC, or xPONENT Software is available on Luminex instruments to analyze your data.

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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.

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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.

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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.

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

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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.

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

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Sorry, the FlowCytomix product line has been discontinued. However, we offer a new antibody and magnetic bead-based detection system, i.e., ProcartaPlex assays for multiplex protein quantitation using the Luminex instrument platform.

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With everything working smoothly, here is the approximate read time for one plate on different Luminex instruments:

- FLEXMAP 3D Systems and INTELLIFLEX Systems will read 96 well plates in ~20 min, and 384 well plates in ~75 min.
- Luminex 100/200 Systems will read 96 well plates in ~40 min.
- MAGPIX System will read 96 well plates in ~50-60 min. Please note that these are estimates for the read times of the plates only, and do not include time for calibration or set-up of the instrument.

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In general, sensitivity between Simplex and Multiplex ProcartaPlex assay kits is comparable.

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We supply the standard protein in a lyophilized form. Lyophilization is a dehydration process that preserves the perishable standard. We have demonstrated that often, the amount of starting protein prior to lyophilization and that of the final product after lyophilization varies (this could be different from analyte to analyte). To ensure that we deliver the most quantitative and reproducible product, we always calibrate the standard after lyophilization and therefore, the concentration range can vary from lot to lot of the standard.

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No. Storage of the beads at temperatures below 0 degrees C will damage the beads and render them unusable.

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We validate and release our ProcartaPlex assays with a minimum bead count of 50. In our experience, the assays also work with a bead count of 20 though we have not validated them at this bead count.

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The sensitivity information is based on the 2 hr incubation with samples.

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To minimize inter-assay coefficient of variation between two independent assay plates, it is very important that the same operator performs the assays. Also, we recommend that you follow the incubation times exactly as they are stated in the manual in order to get consistent results. Other tips for minimizing inter-assay coefficient of variation include performing a standard curve in duplicate on each plate and using the same kit lot.

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In-house, we test ProcartaPlex assays with RPMI + 0.5% FCS. You would have to empirically determine if your specific medium will interfere with the assay.

Note: Our customers have successfully used media containing up to 5% FBS with ProcartaPlex assays. As bovine serum contains TGF beta, the use of FBS or FCS should be avoided with TGF beta ProcartaPlex assays.

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Open the Bio-Plex Manager and click on “File”, then “Document Export Properties”, then “Output CSV Format” and set “Analytes Labels” to “Name (Region)”. Choose the desired file “Destination”, and click “OK”. Click “Document Export”, then “Export”, and import the generated .CSV file into ProcartaPlex Analysis App on Thermo Fisher Connect: https://apps.thermofisher.com/apps/procartaplex

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We have not specifically tested ProcartaPlex assays with tear samples in-house. However, here is a reference from the literature describing the use of ProcartaPlex assays with tear samples (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161422/pdf/pone.0107370.pdf).

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We have tested breast milk samples in-house using the serum ProcartaPlex protocol and found no general interference with the breast milk matrix. Please note that we have only tested a few analytes (TNFa, IL-10, TNFR), however, there is no obvious reason why other analytes should not work as well.

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The size of the beads in our ProcartaPlex assays is 6.5 µm.

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A complete list of available targets for our entire ProtcartaPlex assay portfolio can be found on our website by using the ProcartaPlex Panel Configurator (https://www.thermofisher.com/order/luminex/) and choosing the species of interest. Alternatively, a list of all human ProcartaPlex kits can be pulled up on our website using the search term 'ProcartaPlex Assay Human', or by opening the drop-down menus for Human ProcartaPlex Panels on our ProcartaPlex Assays page (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/luminex-multiplex-assays/procartaplex-immunoassays/procartaplex-assays.html).

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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.

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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.

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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).

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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).

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This may be possible as a custom order. Please reach out to your Sales Representative or to Technical Support (techsupport@thermofisher.com) with your kit number and lot number, they can work with our manufacturing team to determine availability of lot-specific standards.

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Our ProcartaPlex assays have been validated with the Hand-Held Magnetic Plate Washer (Cat. No. EPX-55555-000). We also recommend the Magnetic 96-Well Separator (Cat. No. A14179) as it has a stable magnetic field distributing beads along the bottom of the wells for maximum retention of beads during wash steps.

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If you have a magnetic automated plate washer, this can be used with our magnetic bead kits.

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Yes, ProcartaPlex Streptavidin-PE (Cat. No. EPX-SAPE-000) is available as a stand-alone item. We offer most of our PorcartaPlex buffers and reagents as stand-alone items. A complete list of what is currently available can be found on our website here: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/luminex-multiplex-assays/procartaplex-immunoassays/procartaplex-supplemental-products.html.

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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 here, under "Accessories": https://www.thermofisher.com/us/en/home/life-science/antibodies/immunoassays/procartaplex-assays-luminex/procartaplex-immunoassays/procartaplex-preconfigured-panels.html.

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Typical timeline for sample to results for our ProcartaPlex assays is 4.5 hrs.

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We do list many references for our ProcartaPlex portfolio on our website here: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/luminex-multiplex-assays/procartaplex-immunoassays/procartaplex-immunoassay-publications.html. More recent publications can be found using internet search tools such as Google Scholar or similar.

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We have listed all the species we have tested in our Cross-reactivity Chart for NHP ProcartaPlex Simplex Kits. This chart can be found here (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/luminex-multiplex-assays/procartaplex-immunoassays/procartaplex-assays.html) by clicking on the link for Non-human Primate ProcartaPlex Panels.

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ProcartaPlex High Sensitivity assays are designed to measure small concentration differences in cell culture supernatants, plasma, and serum samples with 10-fold lower LLOQs (Lower Limit of Quantification) and sensitivities for all analytes in the femtogram range. They have been further optimized to overcome the sensitivity detection limits of conventional multiplex immunoassays.

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“Convenience” multiplex panels are pre-mixed and optimized bead sets for use without modification. “Combinable” multiplex panels are target sets which can be combined or supplemented by addition of beads from ProcartaPlex Simplex kits. These two types of panels cannot be multiplexed together.

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Please refer to the assay protocol for kit-specific instructions and your Luminex instrument user guide for instrument-specific instructions. Prior to running the assay, please ensure that the probe height has been calibrated with the 96-Well Flat Bottom Plate supplied with the kit. Failure to adjust the probe height can cause damage to the instrument or low bead count. The Luminex system allows for calibration of low and high RP1 target values. We recommend RP1 low target value settings for immunoassays. Please refer to the lot-specific Certificate of Analysis provided with the kit for bead region and analyte associations when entering the information into the Luminex Acquisition Software.

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Please follow the instructions here (https://tools.thermofisher.com/content/sfs/manuals/Preparation-of-Tissue-Homogenate.pdf).

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We have not tested the use of fixed samples with the ProcartaPlex assay and therefore cannot confirm compatibility at this time.

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Please refer to the lot-specific Certificate of Analysis to check the source of the Standard Mix (i.e., Standard Mix A, B, or C) included with each kit. When combining Simplex Bead Sets with other Simplex Bead Sets or Multiplex kits, multiple vials of the same premixed standard (Standard Mix A, B, or C) may be shipped. If the combination of analytes you want to analyze in your multiplex assay includes two analytes that require the same premixed standard, use only one vial of the premixed standards to prepare the standard curve. If the kits you want to combine include different lots of the same premixed standard, please choose one vial (of any lot) for your multiplex assay.

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You will need to prepare a 7-point standard curve as well as a blank.

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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.

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Yes, you can combine ProcartaPlex Combinable panels and Simplex kits from different orders to run a bigger multiplex experiment. You will want to ensure that there is no overlap of bead regions between the kits you are combining. Please refer to the online ProcartaPlex Panel Configurator (https://www.thermofisher.com/order/luminex).

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No, the Basic Kits are specific to the species listed, i.e. the Mouse Basic Kit can only be used with Mouse Simplex Bead Sets. The Mouse, Human, Canine, Porcine, Rat and NHP ProcartaPlex Basic Kits are not interchangeable.

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When combining a Multiplex kit with Simplex Bead Sets, a Basic kit is not required. All the non-target specific reagents which are included in the Basic kits are components of our Multiplex kits. Only when you are combining multiple Simplex kits (i.e., no Multiplex kit in the panel) will you need to also purchase a Basic kit.

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No. To analyze ProcartaPlex (Luminex) plates, you will need access to a Luminex analyzer such as a Luminex 100/200, MAGPIX, or FLEXMAP 3D system.

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Ideally, we recommend that you analyze the samples immediately. If the plate cannot be read on the day of the assay, cover and store the plate in the dark, overnight 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 (500-600 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.

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To process cell lysates (extract cellular proteins), follow the instructions provided here (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017835_PrepareCellLysates_Procartaplex_PI.pdf).

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Collect spleen, lung, brain, kidney, liver, or heart tissue and treat with or without LPS (100 µg, i.p., for 15 mins, 30 mins, 1 hr, 2 hrs, or 3 hrs). Weigh tissue in a 2 mL microcentrifuge tube. Add 500 µL of Cell Lysis Buffer (Cat. No. EPX-99999-000) per 100 mg of tissue. Add one 5-mm Stainless Steel Bead, then assemble tubes into TissueLyser according to the manufacturer’s recommendations. We recommend using 5-mm Stainless Steel Beads from Qiagen (Cat. No. 69989). Homogenize tissue at 25 Hz for 0.5-3 mins as indicated in the table below. Centrifuge the sample at 16,000 × g for 10 mins at 4 degrees C. Transfer the supernatant to a new microcentrifuge tube. Measure the total protein concentration. Dilute samples to 10 mg protein/mL with 1X PBS. To proceed with ProcartaPlex protocol, add 25 µL of Universal Assay Buffer (Cat. No. EPX-11111-000) to 25 µL of the diluted sample to each sample well.

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