Sample Collection, Dilution and Storage

General Notes and Recommendations:

  • For a Discovery Assay Eve Tech runs all samples in the format in which they are received. If a dilution is required for a Discovery Assay, our clients carry-out the desired dilution prior to sending samples to allow them to save sample volume.  Running a pilot study is recommended to determine the optimal dilution if any is needed.  Please refer to the “Sample Dilution Guide below” below for more details. Running a Custom-plex assay can lead Eve Tech to dilute your samples according to the manufacturer’s protocol or from the results of a pilot study.
  • Eve Technologies includes sample filtration when running assays. Clarifying samples significantly improves results and avoids the majority of sample issues that are caused by cellular debris and other contaminants such as lipids and bacteria.  We have developed an advanced procedure that will dual-stage filter your samples through a Durapore 1.2um filter then a Durapore 0.22um filter with virtually no loss to sample volume and protein available for assay detection.  As a result, you will have better reproducibility and accuracy.
  • ALL samples must still be centrifuged prior to aliquoting. The aliquot should be drawn from the middle of the sample volume.  Samples that have a high amount of contaminants, cellular debris, lipids, or coagulation can result in a volume loss as volume is being displaced by this debris and they can clog the filters.  Highly turbid or coagulated samples that clog the filters will be partially filtered by piercing the clogged membrane.
  • Please do not wrap your tubes in parafilm.
  • Please view our “Tubes, Volumes and Labeling” page
  • We recommend running a pilot assay as a proof of concept to ensure:
  1. Your samples are compatible with the multiplexing bead technology (Milliplex / BioPlex).
  2. Your samples acceptably fall in the standard curve range.
  3. To determine the most optimal dilution required (if any) that will result in the most accurate data.
    • If your research gives an option between serum or plasma, we recommend serum.
    • We recommend running benchmark samples with each assay. Examples: blank media, homogenate buffer, lysis buffer, normal plasma, normal serum.
    • Samples should not contain organic solvents like DMSO. They can negatively affect multiplex assays.
    • To ensure optimal recovery and sensitivity please prepare samples properly and use a consistent protocol.

Serum Preparation:

  • Most rat & mouse cytokine arrays recommend serum be run with a 2-fold dilution. If you are running a Discovery Assay that fits this description, make this dilution using PBS pH~7.5 prior to freezing your samples unless you wish to run your samples undiluted.  By making this dilution you can save sample volume.
  • Allow blood to clot for 30min or more at room temp. After clotting, centrifuge at 1000 x g for 10min at 4°C.
  • If you want your samples run at a dilution, prepare your aliquot accordingly (see Sample Dilution Guide below).
  • Aliquot serum immediately into a pyrogen/endotoxin-free polypropylene tube.
  • Store samples at ≤-20°C (for ~1 month storage life) or ≤-70°C (for more than 1 month storage).
  • Avoid using hemolyzed or lipemic sera.
  • Avoid multiple freeze/thaw cycles (>2 cycles).
  • If assaying for GLP-1, Ghrelin, and/or Amylin do the following:

GLP-1:  Add DPPIV inhibitor

Ghrelin: Add serine protease inhibitor

Amylin:  Add protease Inhibitor cocktail.  Contact us for recommended proteases.

Plasma Preparation:

  • Most rat & mouse cytokine arrays recommend plasma be run with a 2-fold dilution. If you are running a Discovery Assay that fits this description, make this dilution using PBS pH~7.5 prior to freezing your samples unless you wish to run your samples undiluted.  By making this dilution you can save sample volume.
  • Using EDTA as an anticoagulant is recommended for most assays. Avoid using heparin for cytokine arrays.
  • If using heparin as an anticoagulant, do not exceed 10 IU per mL of blood collected. Heparin can cause bead agglutination and can give falsely high results.
  • EDTA or Citrate are not recommended anticoagulants in the Human MMP and TIMP assays due to chelation.
  • Centrifuge at 1000 x g for 10min at 4°C within 30 minutes of blood collection. Repeat centrifuging if necessary.
  • If you want your samples run at a dilution, prepare your aliquot accordingly (see Sample Dilution Guide below).
  • Collect plasma immediately. Aliquot and store samples at ≤-20°C (for ~1 month storage life) or ≤-70°C (for more than 1 month storage).
  • Avoid using hemolyzed or lipemic plasma.
  • To prepare samples for a GLP-1, Ghrelin, and/or Amylin assay, follow protease instructions outlined in serum section.
  • Avoid multiple freeze/thaw cycles (>2 cycles).

Tissue Culture & Cell Culture Supernatant Preparation:

  • Centrifuge samples at 3000 x g prior to aliquoting to remove debris.
  • If you want your samples run at a dilution, prepare your aliquot accordingly (see Sample Dilution Guide below).
  • Transfer supernatant to a new tube and store samples at ≤-20°C or ≤-70°C (for more than 1 month storage).
  • Avoid multiple freeze/thaw cycles (>2 cycles).

Tissue Homogenate Preparation:

  • With the high variation of proteins expressed in different tissues along with the variation in extraction methods, specific analyte detection cannot be guaranteed. Therefore, we recommend running a pilot assay.
  • Homogenize tissue in PBS or equivalent buffer containing protease inhibitors (like aprotinin or an inhibitor cocktail) and low (≤ 0.5%) non-ionic detergent concentration. Avoid using SDS or other denaturing agents. If SDS is required, do not exceed 0.1%.
  • Example buffer: 20 mM Tris HCl (pH 7.5), 0.5% Tween 20, 150 mM NaCl, Sigma protease inhibitors 1:100.
  • RIPA buffer is also a commonly used homogenate buffer for our assays.
  • Once tissues are homogenized Centrifuge at 10,000 x g for 10 minutes at 4° C and transfer the supernatant to a new tube.
  • Samples should be normalized to contain an equal amount of protein. normalize the samples based on total protein by adding buffer to higher total protein samples. Normalizing data is not recommended.  Aim for a minimum protein concentration of 400ug/mL.
  • Store samples at ≤-20°C (for ~1 month storage life) or ≤-70°C (for more than 1 month storage).
  • Avoid multiple freeze/thaw cycles (>2 cycles).

Cell Lysate Preparation:

  • Adherent Cells:
    • Trypsinize and remove cells using a cell scraper. Transfer the cells to a 15 ml conical tube and centrifuge at 500 x g at 4° C for five minutes.
    • Carefully aspirate the supernatant and re-suspend the cells in ice-cold PBS. Repeat this step using ice-cold PBS and centrifuge again at 500 x g at 4° C for five minutes. Completely remove all of the PBS.
  • Suspension Cells:
    • Transfer the cells to a 15 ml conical tube and centrifuge at 500 x g at 4° C for five minutes.
    • Carefully aspirate the supernatant and re-suspend the cells in ice-cold PBS. Repeat this step using ice-cold PBS and centrifuge again at 500 x g at 4° C for 5 minutes. Completely remove all of the PBS.
  • Lyse cells in a PBS-based buffer containing protease inhibitors (like aprotinin or an inhibitor cocktail) and low (< 0.5%) non-ionic detergent concentration.
  • If possible, avoid using SDS or other denaturing agents. If SDS is required, do not use more than 0.1%. Extraction medium should also not contain any organic solvents like DMSO.
  • Example buffer: Tris-HCl: 50 mM, NaCl: 150 mM, 0.5% NP-40, EDTA: 1 mM, Aprotinin: 1mg/ml, pH 7.4.
  • Centrifuge the extract at 10,000 x g for 10 minutes at 4° C.
  • Transfer supernatant to a new polypropylene tube and store samples at ≤-70°C.
  • Avoid multiple freeze/thaw cycles (>2 cycles).

Sputum

  • Remove solid material with high-speed ultracentrifugation. Prior to centrifugation you may need to add a chemical to liquefy viscous samples (ie. hyaluronidase) or add a dilution buffer (PBS pH~7.5) to make a 2-4 fold dilution.
  • Aliquot to a new polypropylene tube and store samples at ≤-70°C.
  • Avoid multiple freeze/thaw cycles (>2 cycles).

BAL fluid

  • Remove solids with centrifugation. You may wish to add 1%BSA in PBS to prevent loss of sample to the labware.
  • Transfer supernatant to a new polypropylene tube and store samples at ≤-70°C.
  • Avoid multiple freeze/thaw cycles (>2 cycles).

Synovial fluid

  • To address the high viscosity of synovial fluid dilute the fluid 4-fold using PBS pH~7.5 or add a chemical to liquefy viscous samples (ie. hyaluronidase).
  • Aliquot to a new polypropylene tube and store samples at ≤-70°C.
  • Avoid multiple freeze/thaw cycles (>2 cycles).

Urine

  • Typically, measurement of analytes in urine requires either a 24 hr urine collection or second morning void collection.  For the second morning void urine it is common to normalize the analyte value is against creatinine (ie. you express your analyte result as units/mg of creatinine).
  • Remove solids with centrifugation.
  • Transfer supernatant to a new polypropylene tube and store samples at ≤-70°C.
  • Avoid multiple freeze/thaw cycles (>2 cycles).
  • Note: EGF detection in human urine requires a single-plex assay run with urine diluted to approx. 1:625.

Other Sample Types:  Please contact us if you have any inquiries about your sample type.

Sample Dilution Guide

The benefit of diluting your own samples prior to shipping to Eve Tech is to lessen the amount of sample volume required – which is particularly important for studies involving mice or rats. Typically most samples can be run undiluted for most assays (particularly cytokine arrays).  However, running a pilot study is recommended to determine if a dilution is required to keep highly concentrated samples within the standard curve limits.  Our standard procedure for a Discovery Assay is to run all samples in the format in which we received them.  Different from a Discovery Assay, a Custom-plex assay can lead Eve Tech to dilute your samples according to the manufacturer’s protocol or from the results of a pilot study.

For our cytokine array Discovery Assays, dilute your samples using standard PBS pH~7.5 prior to shipping for the following cases (refer to Table 1 for details):

  1. You are running serum / plasma on mouse or rat cytokines (unless you want results from undiluted samples).
  1. You are running a pilot study at various dilutions.
  1. You previously know your samples require a dilution. For example your pilot study showed a dilution is required.
Table 1: Required Volumes and Recommended Sample Dilutions
Discovery Assay Volume Required for testing samples in SINGLE *
Volume Required for testing samples in DUPLICATE *
Recommended Dilution Factor
Serum, Plasma, Urine, CSF & BALF Cell Culture Supernatant, Tissue Homogenate & Cell Lysate
Human 64-plex Assay 100µl 150µl Undiluted (Note, PDGF-AA, PDGF-BB, RANTES are high in blood samples and may require a dilution. EGF in urine must be run separate at a dilution) Undiluted (Pilot run recommended)
Human 41-plex or 23-plex Assay 50µl 75µl Undiluted (Note, PDGF-AA, PDGF-BB, RANTES are high in blood samples and may require a dilution. EGF in urine must be run separate at a dilution) Undiluted (Pilot run recommended)
Human MMP & TIMP panel 50µl 75µl Undiluted (Eve Tech makes dilution) Undiluted (Eve Tech makes dilution)
Mouse 32-plex Assay 50µl 75µl 2 fold (25ul sample in 25ul PBS pH~7.5) Undiluted (Pilot run recommended)
Rat 27-plex Assay 50µl 75µl 2 fold (25ul sample in 25ul PBS pH~7.5) Undiluted (Pilot run recommended)
Featured Assays Check website for required volume Check website for required volume Check website for required volume Check website for required volume
Custom-Plex Assays (Pilot study recommended if protocol is not specific with sample type) Inquire Inquire Inquire Inquire

How to Dilute your Samples

  • Dilute your samples in standard PBS pH ~7.5.
  • 2-Fold Dilution:  1 part sample to 1 part buffer (25ul sample to 25ul buffer)
  • 3-Fold Dilution:  1 part sample to 2 parts buffer (17ul sample to 34ul buffer)

 Please Note:

Comparing results from diluted samples to undiluted samples can be inaccurate due to the matrix effect and the inaccurate multiplication of baseline signals.  For more information on the matrix effect, sample homogeneity and other factors that cause inaccuracies when comparing results of different dilution factors, please contact us.