Assay Overview

R&D Systems Rat Cytokine Array Panel A (Catalog # ARY008) is a rapid, sensitive, and economical multiplex array for the simultaneous detection of 29 different cytokines, chemokines, and acute phase proteins in a single sample. Originally developed for chemiluminescent detection, this Proteome Profiler Antibody Array is amenable to Near-infrared (NIR) fluorescence detection using the LI-COR® Odyssey® Infrared Imaging System. To achieve this adaption, the HRP-conjugated Streptavidin provided in the kit is simply replaced with IRDye® 800CW Streptavidin and the arrays are scanned using a LI-COR Odyssey Infrared Imaging System.

An advantage of NIR detection is that protein expression can be quantified over a much wider linear dynamic range compared to that allowed by chemiluminescence methods. This approach facilitates accurate analysis of weak and strong bands on the same blot without the uncertainty and inconvenience of multiple exposures. In addition, NIR does not require film, chemicals for development, or the use of a dark room.

Reagents

Sample Collection & Storage

Avoid repeated freeze-thaw cycles for all sample types.

• Cell Culture Supernates: Remove particulates by centrifugation and assay immediately or aliquot and store samples at < -20 °C.
• Cell Lysates: Rinse cells with PBS, making sure to remove any remaining PBS before adding lysis buffer. Solubilize cells at 1 x 10⁷ cells/mL in lysis buffer. Pipette up and down to resuspend and rock the lysates gently at 2-8 °C for 30 minutes. Microcentrifuge at 14,000 x g for 5 minutes, and transfer the supernate into a clean test tube. Quantitation of sample protein concentrations using a total protein assay is recommended. For the initial experiment, use a quantity of lysate similar to that used for Western blot. Use the lysates immediately or aliquot and store at < -70 °C.
• Serum: Allow blood samples to clot for 2 hours at room temperature before centrifuging for 20 minutes at 2000 x g. Remove serum and assay immediately or aliquot and store samples at < -20 °C.
• Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge for 15 minutes at approximately 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at < -20 °C.
• Tissue Lysates: Excise tissue and homogenize in PBS with protease inhibitors. Add Triton® X-100 to a final concentration of 1%. Freeze samples at < -70 °C, thaw, and centrifuge at 10,000 x g for 5 minutes to remove cellular debris. Quantitation of sample protein concentrations using a total protein assay is recommended. Assay immediately or aliquot and store samples at < -70 °C.

Reagent Preparation

Bring all reagents to room temperature before use.

• Rat Cytokine Array Panel A: Four nitrocellulose membranes each containing 29 different cytokine antibodies printed in duplicate. Handle arrays only with gloved hands and flat-tipped tweezers. After opening, reseal unused membranes in the foil pouch with desiccant and store at 2-8 °C for up to 3 months.*
• Detection Antibody Cocktail: One vial of lyophilized biotinylated antibodies. Before use, reconstitute the Detection Antibody Cocktail with 100 µL of deionized or distilled water. Store at 2-8 °C for up to 3 months after reconstitution.*
• Array Buffer 4: Ready for use. Store at 2-8 °C for up to 3 months after initial use.* May contain a precipitate. Mix well before and during use.
• Array Buffer 6: Ready for use. Store at 2-8 °C for up to 3 months after initial use.*
• 1X Wash Buffer: Dilute 40 mL of 25X Wash Buffer Concentrate in 960 mL of deionized or distilled water. Store at 2-8 °C for up to 3 months after initial use.*

*Provided this is within the expiration date of the kit.

Assay Procedure

Keep samples on ice. To avoid contamination, wear gloves while performing the procedures.

1. Prepare all reagents and samples as directed in the previous sections.
2. Pipette 2.0 mL of Array Buffer 6 into each well of the 4-Well Multi-dish to be used.
3. Using flat-tip tweezers, remove each membrane to be used from between the protective sheets.  Carefully cut off the stamped identification number on the membrane using scissors.  Membrane may be labeled with pencil in the empty space to the right side of the array.  Pick up the membrane from this edge during all processing steps.
4. Place one membrane in each well of the 4-Well Multi-Dish.

   Note: Upon contact with Array Buffer 6, the blue dye from the spots will disappear, but the capture antibodies are retained in their specific locations.

5. Incubate for one hour on a rocking platform. Orient the tray so that each array rocks end to end in its well. Array Buffer 6 serves as a block buffer.
6. While the membranes are blocking, prepare samples by adding up to 1 mL of each sample to 0.5 mL of Array Buffer 4 in separate tubes. Adjust to a final volume of 1.5 mL with Array Buffer 5 as necessary.
7. Add 15 µL of reconstituted Rat Cytokine Array Detection Antibody Cocktail to each prepared sample. Mix and incubate at room temperature for one hour.
8. Aspirate Array Buffer 6 from the wells of the 4-Well Multi-dish and add sample/antibody mixtures prepared in step 7. Place the lid on the 4-Well Multi-dish.
9. Incubate overnight at 2-8 °C on a rocking platform.

   Note: A shorter incubation time may be used if optimal sensitivity is not required.

10. Carefully remove each membrane and place into individual plastic containers with 20 mL of 1X Wash Buffer. Rinse the 4-Well Multi-dish with deionized or distilled water and dry thoroughly.
11. Wash each membrane with 1X Wash Buffer for 10 minutes on a rocking platform shaker. Repeat two times for a total of three washes.
12. Dilute the IRDye 800CW Streptavidin (LI-COR, Catalog # 926-32230) 1:2000 in Array Buffer 6. Pipette 1.5 mL of diluted IRDye 800CW Streptavidin into each well of the 4-Well Multi-Dish.
13. Carefully remove each membrane from its wash container. Allow excess buffer to drain from the membrane by blotting the lower edge onto absorbent paper. Return the membrane to the 4-Well Multi-dish containing the diluted IRDye 800CW Streptavidin. Cover the wells with the lid.
14. Incubate for 30 minutes at room temperature on a rocking platform.
15. Wash each array as described in steps 9 and 10.
16. Carefully remove each membrane from the wash container. Allow excess Wash Buffer to drain from the membrane by blotting the lower edge onto absorbent paper. Collect images with an Odyssey Imager.

Data Analysis

The positive signals seen on developed film can be quickly identified by placing the transparency overlay on the array image and aligning it with the three pairs of positive control spots in the corners of each array. The location of controls and cytokine capture antibodies is listed in the Appendix of the product insert.

Suggested starting Odyssey scan parameters:

Resolution: 84 µm
Quality: Medium
Focus offset: 0.0 mm
Intensity: 5, adjust as necessary.

Collected images may be manipulated using Adjust Image Curve and Alter Image Display Odyssey settings.

Technical Hints & Limitations

• For research use only. Not for use in diagnostic procedures.
• This kit should not be used beyond the expiration date on the kit label.
• Do not mix or substitute reagents with those from other lots or sources.
• Any variation in buffers, operator, pipetting technique, washing technique, and incubation time or temperature can alter the performance of the kit.
• These Human Cytokine Array Panel A membranes are validated for single use only.
• Always use gloved hands and flat-tipped tweezers to handle the membranes.
• A thorough and consistent wash technique is essential for proper assay performance.
• Wash Buffer should be removed completely from the membrane before proceeding to the next step.
• Avoid microbial contamination of reagents and buffers.
• Soluble receptors and other proteins present in biological samples do not necessarily interfere with the measurement of cytokines in samples. However, until these proteins have been tested with the Proteome Profiler Array, the possibility of interference cannot be excluded.
• Stamped identification number on membranes must be removed carefully with scissors prior to block step.
• Membrane may be labeled with pencil in the empty space to the right side of the array.  Pick up the membrane from this edge during all processing steps.
• Do not allow the membranes to dry out during any step of the protocol.  Membranes should be scanned wet and bubbles should be carefully removed when placing membranes on the Odyssey scanning bed.  

Data Examples

Figure 1. The Rat Cytokine Array detects multiple analytes in cell culture supernates and lysates.  A. and B. Supernates from NR8383 rat alveolar macrophage cell line untreated or treated with 50 ng/mL LPS for 24 hours. 500 µL of supernate was used for each array shown. C. and D. Lysates from NR8383 rat alveolar macrophage cell line untreated or treated with 50 ng/mL LPS for 24 hours. 200 µg of lysates was used for each array shown. Array images were collected and analyzed using the LI-COR Odyssey Infrared Imaging System.

Figure 2. The Rat Cytokine Array detects cytokines present in serum and tissue. Representative membranes and selective quantification are shown for rat serum (A., 150 µL), lysates of rat lung tissue (B., 400 µg), and lysates of rat liver tissue (C., 400 µg). Array images were collected and analyzed using the LI-COR Odyssey Infrared Imaging System.

LI-COR, Odyssey, and IRDye are registered trademarks of LI-COR, Inc.
Triton is a registered trademark of Union Carbide.