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.

• General Sample Preparation and Handling Tips
• Serum Preparation
• Plasma Preparation
• Cell Culture Supernatant Preparation
• Peripheral Blood Mononuclear Cell (PBMC) Preparation
  • Short Protocol for PBMCs
• Urine Preparation
• MILLIPLEX^® Kits Requiring Special Sample Preparation
• Protocols Using Other Sample Types
• Request Information

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.

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.

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

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For Research Use Only. Not For Use In Diagnostic Procedures.