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HomeMILLIPLEX® Multiplex for Luminex® ImmunoassaysTips & Tricks: Sample Handling and Preparation for MILLIPLEX® Multiplex Panels

Tips & Tricks: Sample Handling and Preparation for MILLIPLEX® Multiplex Panels

Proper sample handling and preparation are essential for running immunoassays. From sample collection and storage, to sample dilution requirements, each step is critical to achieving the best results for your research. Most MILLIPLEX® kits have been tested for serum, plasma, and cell culture supernatant, and this quick guide outlines best practices for preparing these sample types for use in MILLIPLEX® multiplex assays.

Section Overview

Watch this video featuring one of our expert technical support scientists for advice on preparing and handling samples for use in MILLIPLEX® multiplex assays.

General Sample Preparation and Handling Tips

  • Avoid multiple freeze/thaw cycles on your samples.
  • Analyte concentrations may differ between serum and plasma. Be consistent with the use of sample type within a study.
    • If you have the option of using serum or plasma, we recommend you choose serum as it tends to be cleaner.
  • All samples must be stored in polypropylene tubes. Do not store samples in glass.
  • Vortexing is recommended for homogeneous sample prep.
  • Proper and consistent pipetting technique is key to accurate data, especially if multiple users will be generating data in collaboration.
    • Maintaining properly calibrated pipettors is important, and training on best practices for pipetting can substantially increase pipetting precision.
    • Use reverse-pipetting for more accurate dispensing. Refer to the video at the top of the page to learn this technique.
  • Follow MILLIPLEX® protocol instructions for sample handling and dilutions.
    • Always read the entire protocol before proceeding. Procedures are optimized for best data results and can vary from kit to kit.
    • Some MILLIPLEX® kits for metabolic biomarkers require addition of protease and/or phosphatase inhibitors to samples.
    • Other MILLIPLEX® kits may require sample extraction or acidification.

Serum Preparation

  • Serum separator tubes (SST) are recommended for higher-quality separation.
  • Allow the blood to clot for at least 30 minutes before centrifugation for 10 minutes at 1,000 x g to separate cells.
  • Remove serum and run your assay immediately or aliquot and store samples at -20° to -80°C.
  • Hemolysis can result in increased proteolytic activity and analyte degradation primarily due to enzymes released from lysed cells.
    • Trace hemolysis in samples collected with protease inhibitors may be acceptable, but gross hemolysis will likely interfere with assay performance.
    • If you must use serum with gross hemolysis or lipemia, avoid debris, lipids, and cells.
  • Hemoglobin (at >10 mg/mL) is known to interfere with antigen/antibody interactions.
  • Dilute samples according to the specific MILLIPLEX® kit protocol.

Plasma Preparation

  • Plasma collection using EDTA as an anticoagulant is recommended.
  • Care must be taken when using heparin as an anticoagulant since an excess of heparin will provide falsely high values.
    • Use no more than 10 IU heparin per mL of blood collected.
  • Other anticoagulants have not been extensively tested and are not recommended.
  • Centrifuge for 10 minutes at 1,000 x g within 30 minutes of blood collection.
  • Remove plasma and assay immediately or aliquot and store samples at -20° to -80°C.
  • Dilute samples according to the specific MILLIPLEX® kit protocol.

Cell Culture Supernatant Preparation

  • Centrifuge samples to remove debris and assay immediately or aliquot and store samples at -20°C to -80°C.
  • For cell culture supernatants, use fresh culture medium as the matrix solution in the blank, standard curves, and controls.
  • If cell culture medium is used as matrix solution, be certain there are no active proteases, phosphatases, or supplements present which may interfere with the assay or generate inaccurate results (e.g., cytokines, human serum, etc.).
  • If samples are diluted in assay buffer, use the assay buffer as the matrix.
  • Some kits may have specific requirements, always refer to the kit protocol before planning your experiment.

Peripheral Blood Mononuclear Cell (PBMC) Preparation

Note: PBMC sample prep is the most critical step for obtaining reproducible results.

  • Strong detergents are used in lysis buffer. Enough detergent in the lysis buffer is required to solubilize proteins. Do not exceed total protein concentrations of 5-6 mg/mL.
    • A drop in signal has been observed for several analytes using PBMC samples at >6 mg/mL total protein (not enough lysis buffer was added to solubilize proteins).
  • Because strong detergents are used in the lysis buffer, lysate samples require enough dilution in assay buffer to dilute strong detergents. Avoid lysate total protein concentrations below 2 mg/mL.
    • If lysate protein concentration is below 2 mg/mL, then too much lysis buffer, with strong detergents, will be present in the assay and will result in decreased signal.
    • If protein concentration below 2 mg/mL is unavoidable, we recommend running less sample, thus minimizing the volume of lysis buffer present in the assay.
  • The optimal total protein concentration is 2-6 mg/mL. Using PBMCs, we determined that 10 μL of lysis buffer per 1 million PBMC cells yields approximately 2 mg/mL. Adding 10 μL of this 2 mg/mL sample plus 15 μL of assay buffer yielded good results. As a starting point, it is recommended to add 10 μL of lysis buffer per 2 million PBMC cells.
    • Never dilute samples in lysis buffer, rather dilute in assay buffer which does not contain strong detergents.

Short Protocol for PBMCs

  1. If PBMCs are from frozen stock, it is recommended to allow cells to recover 24 hours in complete media. (Less than 24 hours recovery leads to decrease in signal.) After 24 hours of recovery, count cells using an appropriate cell counter.
  2. Pellet the PBMCs at 1,000 x g using a tabletop centrifuge for 5 minutes at room temperature.
  3. Remove supernatant and wash cells with PBS.
  4. Pellet the PBMCs at 1,000 x g using a tabletop centrifuge for 5 minutes at room temperature.
  5. Remove wash buffer and add 10 μL lysis buffer (with 2x concentrated protease inhibitors added just prior to use) per 2 million cells.
  6. Gently vortex for 30 seconds before transferring cell lysate into a centrifuge tube.
  7. Gently rock cell lysate for 10 minutes at 4°C. Pellet unbroken cells and organelles at 12,000 x g for 10 minutes at 4°C.
  8. Transfer clear supernatant into a new centrifuge tube.
  9. It is recommended, at least for the first time, to determine total protein concentration. If not, then it is recommended to run a lysate titration starting at 10 μL sample + 15 μL of assay buffer 1 and performing a 1:1 serial dilution in assay buffer 1. Add protease inhibitors (such as Protease Inhibitor Cocktail I or AEBSF) and/or phosphatase inhibitors to “home-brew” lysis buffers.

For more details on total protein concentration and MILLIPLEX® Lysis Buffers, download our Tips & Tricks brochure.

Urine Preparation

Typically, measurement of analytes in urine requires either a 24-hour urine collection or second morning void collection. For the second morning void urine, the analyte value is normalized against creatinine, i.e., the analyte is expressed as units/mg of creatinine.

  • Centrifuge the samples briefly to pellet debris.
  • When using frozen samples, it is recommended to thaw the samples completely, mix well by vortexing, and centrifuge prior to use in the assay to remove particulates.
  • An optimal dilution factor for samples needs to be determined. Assay Buffer provided in the kit should be used as the sample diluent.

MILLIPLEX® Kits Requiring Special Sample Preparation

Table 1 describes the special sample preparation required for certain MILLIPLEX® multiplex kits.

Kit NameProduct No.Sample Type Requiring PreparationSample Treatment/ InhibitorsInhibitor Source
Canine Gut Hormone PanelCGTMAG-98KSerum, PlasmaDPP-IV, Aprotinin, AEBSF, Protease Inhibitor CocktailSee Notes 1,2,3,4
Human Metabolic Hormone PanelHMHEMAG-34KSerum, PlasmaDPP-IV, Aprotinin, AEBSF, Protease Inhibitor CocktailSee Notes 1,2,3,4,5
Human Metabolic Hormone V3 PanelHMH3-34KSerum, PlasmaDPP-IV, Aprotinin, AEBSF, Protease Inhibitor CocktailSee Notes 1,2,3,4,5
Mouse Metabolic Hormone PanelMMHE-44KSerum, PlasmaDPP-IV, Aprotinin, AEBSF, Protease Inhibitor CocktailSee Notes 1,2,3,4,5
Non-Human Primate Metabolic PanelNHPMHMAG-45KSerum, PlasmaDPP-IV, Aprotinin, AEBSF, Protease Inhibitor CocktailSee Notes 1,2,3,4
Rat Metabolic Hormone PanelRMHMAG-84KSerum, PlasmaDPP-IV, Aprotinin, AEBSF, Protease Inhibitor CocktailSee Notes 1,2,3,4
Human IGF PanelHIGFMAG-52KSerum, PlasmaExtractionNone
Human IGF Binding Protein PanelHIGFBMAG-53KSerum, PlasmaProtease Inhibitor CocktailSee Note 4
Human Neuropeptide PanelHNPMAG-35KSerum, PlasmaAcetonitrile ExtractionNone
Rat/Mouse Neuropeptide PanelRMNPMAG-83KSerum, PlasmaExtractionNone
Multi-Species Hormone PanelMSHMAG-21KSerum, PlasmaAcetonitrile ExtractionNone
Human Skin PanelSKINMAG-50KSkin LysatesCentrifuge to Remove DebrisNone
Multi-Species TGFβ1 Singleplex PanelTGFBMAG-64K-01Serum, Plasma, CCSAcidificationNone
Multi-Species TGFβ1, 2, 3 PanelTGFBMAG-64K-03Serum, Plasma, CCSAcidificationNone
Human Circadian Stress PanelHNCSMAG-35KSerum, PlasmaAcetonitrile ExtractionNone

Notes

1. DPP-IV (Product No. DPP4-010) is used at 10 µL per mL of blood.
2. Pefabloc or AEBSF (Product No. 101500) is used at 1 mg/mL in blood.
3. Protease Inhibitor Cocktail I (Product No. 20-201).
4. Protease Inhibitor Cocktail (Product No. P2714).
5. Active and Total cannot be run together in the same assay.

Table 1. MILLIPLEX® kits requiring special sample preparation including the specific sample type and treatment/inhibitor.

Protocols Using Other Sample Types

Protocols are available for sample types beyond what has been tested in MILLIPLEX® kits. Some examples are described in Table 2.

Sample TypeSpeciesProtocolReference (if applicable)
Bronchoalveolar Lavage (BAL) For lavage samples, use 50 μL sample + 25 μL beads in sample wells. Set up standards using one additional lower point and dropping the highest concentration standard point. Use a buffer matrix or medium used to collect the lavage sample as the matrix, i.e., 25 μL standard/ control/blank + 25 μL assay buffer /medium + 25 μL beads. The first incubation with standard/sample should be overnight at 4°C. Results should be divided by 2. 
Infectious Samples For infectious samples: If washing with an automatic plate washer, add 30% bleach to the waste bottle before washing/aspirating the plate. If washing with a handheld magnetic bead separator, add 30% bleach to a container capable of catching the wash solution decanted from the plate. Then at the end of the assay, resuspend the beads in 0.1 mL of 4% formaldehyde made in 10 mM PBS (prepared fresh daily) instead of sheath fluid, before running the plate in the Luminex® machine. Prolonged incubation in this solution may cause bead aggregation. Consequently, after agitating the plate for 5 minutes on an orbital plate shaker, read the plate immediately. 
Lipemic Samples For lipemic and plasma samples, the blood needs to be collected on ice, centrifuged in a refrigerated centrifuge, aliquoted, and frozen at -20°C for short term (<2 months) and -70°C for long term. Prior to assay setup, thaw samples and centrifuge at 10,000 rpm for 5 minutes. Spool off the lipid layer from the surface using a cotton swab and use the supernatant below lipid layer for the assay. 
SputumHumanFresh, spontaneously expectorated sputum samples were frozen and stored at − 80°C until preparation. Samples were ultra-centrifuged for 4 hours at 38,000 rpm.Hansen CR, et al. “Inflammation in Achromobacter xylosoxidans infected cystic fibrosis patients.” Journal of Cystic Fibrosis vol. 9 51-58. 2010. DOI: 10.1016/j.jcf.2009.10.005
SalivaHumanAdd protease inhibitor cocktail at 1:500 to saliva. Centrifuge at 10,000 rpm for 10 minutes and dilute supernatant 1:2 with assay buffer prior to assay setup. This method significantly improves recovery and reduces bead aggregation. Run assay with assay buffer as matrix in standard curve. Use an overnight option if available. 
Cell or Tissue Extraction Protocol varies depending on tissue types and/or analytes of interest. Generally, most protocols that are used in ELISAs can be used, but here are some guidelines for selecting a method.

1) Homogenize cells or tissues mechanically (e.g., ultrasonication) in a PBS-based buffer containing protease inhibitors (like aprotinin or an inhibitor cocktail) and low (< 0.2%) non-ionic detergent concentration.

2) Extraction medium should not contain any organic solvents like DMSO, etc.

3) Centrifuge the extract and freeze supernatant at <-20°C.

4) Use the extraction medium as matrix in blank, standard curve, and QCs.		 
TearsHumanUnstimulated tear samples were collected non-traumatically from the external canthus of open eyes, avoiding additional tear reflex as much as possible. Glass capillary micropipettes (Drummond, Broomall, PA) were used to collect 1 μL of tears. Each sample was then diluted 1:10 in a sterile collection tube containing ice-cold assay buffer. Tubes with tear samples were kept cold (4°C) during collection and stored at −80°C until assayed. The samples were obtained from right eye (OD) and left eye (OS) of each individual and were not pooled.Enríquez-de-Salamanca, Amalia et al. “Tear cytokine and chemokine analysis and clinical correlations in evaporative-type dry eye disease.” Molecular vision vol. 16 862-73. 19 May. 2010
Gingival Crevicular Fluid (GCF)HumanGCF samples were collected from each site with paper filter strips (PerioPaper) gently inserted into the sites 1 to 2 mm for ~10 sec. Volume of GCF was calculated with a micro-moisture meter (Periotron 8000) and through calibration curves. Samples were frozen at −80°C until analyses of soluble biomarkers were performed.Branco-de-Almeida LS, et al. “Local and Plasma Biomarker Profiles in Localized Aggressive Periodontitis.” JDR Clin Trans Res. Jul;2(3):258-268. 2017. DOI: 10.1177/2380084417701898
Synovial Fluid (SF)HumanAdd an additional standard point compared to standard protocol in a buffer matrix. Use 100 μL buffer + 25 μL std/sample. Agitate for 10 min at room temperature. Add 25 μL beads and incubate with agitation overnight at 4°C. On the next day, before reading, resuspend the beads in 200 μL of 1X wash buffer prior to loading onto the instrument. Volume read is 100 μL on the instrument. 
Synovial Fluid (SF)EquineSynovial fluid (2 mL) was aseptically collected and aliquoted (EDTA and Protein LoBind microfuge tubes, Eppendorf® Labware, Westbury, CT). Anticoagulant-free synovial fluid was immediately centrifuged at 12,000 × g for 10 min at 4°C and the supernatant stored at −20°C. Thawed samples (200 μL) were hyaluronidase-digested (10 μL of 100 IU hyaluronidase/mL acetate buffer; Worthington Biochemical Corporation, Lakewood, NJ) for 30 min at 37°C, centrifuged at 12,000 × g for 10 min at 4°C, and the supernatant recovered.Front. Vet. Sci., 7:568756. 26 November 2020. DOI: 10.3389/fvets.2020.568756
Fecal SamplesMouseFecal contents from small and large intestines were collected when mice were euthanized and were frozen at −80°C. Samples were thawed out, weighed, and resuspended in PBS 5% gelatin supplemented with complete protease inhibitor cocktail (Roche), with the help of vigorous vortexing. Samples were prepared as concentrated as possible to facilitate detection of low abounding cytokines. Insoluble fraction was separated by centrifugation at 10,000 × g for 10 min at 4°C, in an Eppendorf® 5417R centrifuge. Supernatants were either tested immediately or frozen at −80°C.Toxicology and Applied Pharmacology, Volume 333, 2017, Pages 84-91, ISSN 0041-008X, DOI: 10.1016/j.taap.2017.08.013
Peritoneal FluidMouseAfter animals were euthanized, 1 mL of sterile PBS was injected into the peritoneal cavity, the abdominal area was gently massaged, and the fluid was collected. The collected fluid was centrifuged at 1,390 rpm for 5 min at 4°C and the resulting supernatant was then stored at −80°C.Ruiz A, et al. Effect of hydroxychloroquine and characterization of autophagy in a mouse model of endometriosis. Cell Death Dis. 2016 Jan 14;7(1):e2059. DOI: 10.1038/cddis.2015.361.
Dried Blood SpotsHumanFinger-stick capillary blood samples were collected on Whatman 903 Protein Saver Card (Maidstone, U.K.) and frozen with desiccant in sealed bags prior to elution. DBS samples (2, 6 mm punches) were eluted and transferred through 96-well Multiscreen® and Ultracel® plates using elution buffer (PBS with 0.5 M NaCl and 0.1% Tween-20) for 16-18 hours in the refrigerator. The resultant DBS elute was resuspended in 90 mL of nuclease-free water.Prado, EA, et al. "Using Dry Blood Spots to Evaluate Serum Cytokines and Chemokines in Humans via Multiplex Technology," International Journal of Exercise Science: Conference Proceedings: Vol. 2: Iss. 6, Article 12. 2014
Table 2.Examples of protocols for other sample types than what is listed in MILLIPLEX® multiplex kit protocols.

For the full list of sample type information for MILLIPLEX® panels, see Appendix 3 in our Tips & Tricks brochure.

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