Most Q-PlexTM Arrays are validated for use with serum and plasma, but for those who want to test other sample types, we offer:
- FREE Demo Kit or FREE in-house sample testing of four samples: This provides the user with an opportunity to screen several sample preparation conditions and see how they work with standard Q-PlexTM assay conditions. In fact, the cytokine levels of a wide variety of tissue homogenates and cell lysates, such as mouse lung, liver, kidney, pancreas, kidney, and eye have been successfully analyzed using Q-PlexTM Arrays under standard assay conditions.
- Custom development: Quansys can perform optimization and validation of assay conditions specific for the user’s sample type.
Preparing Tissue Homogenates and Cell Lysates for use in Q-PlexTM Arrays
The cytokine levels of a wide variety of tissue homogenates and cell lysates, including mouse lung, liver, kidney, pancreas, kidney, and eye, have been successfully measured using Q-PlexTM Arrays. Most homogenization or lysis protocols for an ELISA or western blot are fine to try with Q-PlexTM Arrays as long as the procedure is standardized across samples, the samples are kept cold at all times, and the diluents do not contain chemicals that interfere with the assay. Use the example protocols and reagent compatibility table below as a guideline in designing the optimal homogenization protocol for your sample type.
General Protocol for Tissue Homogenization/Cell Lysate Preparation for Q-PlexTM Arrays: KEEP SAMPLES COLD AT ALL TIMES
- Add ice-cold sterile non-denaturing medium to samples at 10-20% tissue weight/total volume. A typical medium is PBS containing a protease inhibitor cocktail (leupeptin, aprotinin, pepstatin, etc.). Cell lysates must have protease inhibitors, and may also benefit from low levels of chemicals that promote lysis (salts or sugars to create a hypotonic solution, detergents, etc.) as well as DNase (25-50 µg/mL) and RNase (50 µg/mL) to reduce the viscosity increase caused by the release of nucleic acids.
- Homogenize diluted tissues using pre-cooled equipment such as a manual or mechanical tissue homogenizer, mortar and pestle, sonication, etc. In cases where homogenization but not cell lysis is desired, e.g. only intercellular cytokines and not cytoplasmic cytokines are meant to be measured, use an extra gentle homogenization protocol, such as placing tissue and media in a sealable plastic bag and rolling a cylindrical object back and forth over the bag.
- Separate insoluble debris by centrifugation at 4oC. Spinning samples at 10,000 x g for 10-20 minutes is typical. For additional clarity, centrifuge again or filter using a low-protein-binding 4.5 micron filter.
- Keep clarified supernatant to be assayed right away at 4oC, or aliquot and store at -80oC. Minimize freeze/thaw cycles.
- Measure total protein levels of each sample before or after the ELISA so that cytokine data can be normalized to negate differences due to sample collection.The measurement can be done by spectrophotometric absorbance analysis at 280 nm, or by such methods as a Bradford or BCA assay. ELISA results are normalized by dividing the cytokine testing results (in pg/mL) by the total protein content (in micrograms or milligrams/mL) to obtain pg of cytokines/microgram of tissue.
- Assay clarified supernatants to determine cytokine levels. Dilute samples at least 1:2 (parts:total) in the Q-PlexTM Array Sample Diluent to avoid false positives.
To view an example of a published protocol that has been successful for many of our customers homogenizing a variety of tissues, see: Clin Exp Immunol. 2004 July; 137(1): 65–73:
Compatibility of Common Medium Components:Avoid preparing samples using regents that interfere with the assay.
|NP40||No interference at 1%|
|Tween||No interference at 1%|
|Triton||No interference at 1%|
|Citrate||No interference at 20%|
|EDTA||No interference at 20 mM|
|Water||No interference at 55 M|
|Heparin||No interference at 30 mg/ml|
|Urea||No interference at 1 M|
|DMEM||No interference at 100%|
|HAMS||No interference at 100%|
|RPMI||No interference at 100%|
|SFM4||No interference at 100%|