Sample Preparation Guide

General Notes and Recommendations:

NOTE: Conforming tubes are .5 – .65 mL snap cap tubes and label alpha numeric, consecutively, i.e. S1, S2, S3, etc. (S = first letter of your last name).  Non-conforming tubes and or labels are $3.00 per tube.

*Please DO NOT use Parafilm or tape to seal your tubes. This is non-conforming

*Using labels or stickers is NOT recommended – labels can come off when frozen.

• 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
• Samples will be kept for 1 calendar month post analysis and then disposed of unless sample storage is stipulated and paid for on your order.

• 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.  If using heparin as an anticoagulant, do not exceed 10 IU per mL of blood collected.
    • Heparin has also been reported to cause falsely high results in certain assay platforms and analytes.
    • 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.
    • 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.
    • 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:

• • Use a PBS-based buffer containing protease inhibitors.  We recommend using the Sigma Aldrich Protease Inhibitor Cocktail (P2714).

    Use non-ionic detergents, such as NP40, in experimental design.

    Avoid using SDS or other denaturing agents. If SDS is required, do not use more than 0.1%.  Avoid any organic solvents like DMSO*.

    Example buffers:

    Tris-HCl: 50 mM, NaCl: 150 mM, 0.5% NP-40, EDTA: 1 mM, Aprotinin: 1mg/ml, pH 7.4.

    RIPA is a commonly used buffer in Luminex assay performance

    Ultimately, for cell/tissue homogenates, the final cell or tissue homogenate should be prepared in a buffer that has a neutral pH, contains minimal detergents or strongly denaturing agents and has an ionic strength close to physiological concentrations.

     *DMSO is a commonly used polar aprotic solvent.  Although DMSO is not recommended by our preferred kit supplier, they see no negative effect up to 10% DMSO.  If using DMSO, and if you anticipate levels to exceed this, consider a more rigorous washing before the lysis step.

    We recommend including a media control in your sample submission, to understand any background baseline signal associated with your media.

• • Non-adherent cells:
    1. Pellet cells by low-speed centrifugation (500 x g) for 5 minutes.
    2. Remove medium from the pellet, and wash twice with ice cold PBS.
    3. Remove the PBS, and resuspend the cell pellet in cell lysis buffer (recommended cell lysate concentration is 2–5 mg/ml). Incubate 15 minutes on ice with occasional vortexing.
    4. Transfer the lysate to a microcentrifuge tube and centrifuge at 10,000 x g for 10 minutes at 2–8°C to remove particulate matter.
    5. Aliquot the cleared lysate into clean microcentrifuge tubes and determine total protein concentration.

    Adherent cells:

    1. Remove tissue culture medium from the cells, and wash cells twice with ice cold PBS.
    2. Remove the PBS, add cell lysis buffer (recommended cell lysate concentration is 2 to 5 mg/ml), and incubate 15 minutes on ice.
    3. Collect the cell lysate and transfer to a microcentrifuge tube. Centrifuge at 10,000 x g for 10 minutes at 2–8°C to remove particulate matter.
    4. Aliquot the cleared lysate into clean microcentrifuge tubes and determine total protein concentration.

    Lysates should be frozen and stored/shipped at –80°C. Avoid multiple freeze-thaw cycles of frozen samples.

Sputum

• Remove solid material with high-speed ultracentrifugation.
• Aliquot to a new polypropylene tube and store samples at ≤-70°C.
• Avoid multiple freeze/thaw cycles (>2 cycles).
• * Consider Mucolyse treatment for highly viscous samples – please contact Eve Technologies for more information

BAL fluid

• Remove solids with centrifugation. You may wish to add 1%BSA in PBS to prevent loss of sample to the labware.
• * Consider Mucolyse treatment for highly viscous samples – please contact Eve Technologies for more information
• 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:

1. You are running serum / plasma on mouse or rat cytokines (unless you want results from undiluted samples).
2. You are running a pilot study at various dilutions.
3. You previously know your samples require a dilution. For example your pilot study showed a dilution is required.

 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.