Protein analysis

For the analysis of the protein arrays, we did not set any threshold to exclude any potentially differentially expressed cytokines/chemokines in the array. Hence, the signals detected on Image J were recorded and shown. As listed in the supplemental materials, many of the signals were indeed close to zero. We validated any of the candidate cytokines/chemokines using ELISA. We are attaching a detailed protocol here for your further notice.

Protein analysis (Cytokine array) protocol

1. Tissue lysate/serum preparation

a. tissue lysate

• Excise tissue and homogenize in 0.1%Tween/PBS with protease inhibitor.  
• Freeze samples at ≤ -70 °C, thaw, and centrifuge at 10,000 x g for 5 min to remove cellular debris.
• Sonicate for 1 min and centrifuge at 13,000 x g for 10 min to collect supernatant.
• Assay immediately or aliquot and store at ≤ -70 °C.

b. serum

• Allow blood samples to clot for 2 hours at room temperature before centrifuging for 15 minutes at 2000 x g.
• Remove serum and assay immediately or aliquot and store samples at ≤ -20 °C. 

2. Cytokine assay (ARY028, R&D systems)

• Place cytokine array membranes in the 4-Well Multi-dish, and pipet 2.0 mL of Array Buffer 6 into each separate well, then incubate for one hour on a rocking platform shaker
• While the arrays 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 6 as necessary.
• Aspirate Array Buffer 6 from the wells of the 4-Well Multi-dish and add the prepared samples. Incubate overnight at 2-8 °C on a rocking platform shaker.
• Wash each membrane with 1X Wash Buffer for 10 min on a rocking platform shaker. Repeat two times for a total of three washes.
• For each array, add 30 μL of Detection Antibody Cocktail to 1.5 mL of 1X Array Buffer 4/6, and incubate for 1 hour at room temperature on a rocking platform shaker. 
• Wash each array as described above, and pipette 2.0 mL of 1X Streptavidin-HRP into each well of the 4-Well Multi-dish.
• Incubate for 30 minutes at room temperature on a rocking platform shaker. 
• Wash each array as described above, and pipette 1.0 mL of the prepared Chemi Reagent Mix evenly onto each membrane. 
• Carefully cover with the top sheet of the plastic sheet protector. Gently smooth out any air bubbles and ensure Chemi Reagent Mix is spread evenly to all corners of each membrane. Incubate for 1 min.
• Position paper towels on the top and sides of the plastic sheet protector containing the membranes and carefully squeeze out excess Chemi Reagent Mix.
• Leaving membranes on the bottom plastic sheet protector, cover the membranes with plastic wrap taking care to gently smooth out any air bubbles. Wrap the excess plastic wrap around the back of the sheet protector so that the membranes and sheet protector are completely wrapped.
• Place the membranes with the identification numbers facing up in an autoradiography film cassette.
• Expose membranes to X-ray film for 1-10 minutes. Multiple exposure times are recommended.

3. Data analysis

• Pixel densities on developed X-ray film can be collected and analyzed using a transmission mode scanner and Image J software.
• Create a template to analyze pixel density in each spot of the array.
• Export signal values to a spreadsheet file for manipulation in a program such as Microsoft Excel.
• Determine the average signal (pixel density) of the pair of duplicate spots representing each analyte.
• Subtract an averaged background signal from each spot. Use a signal from a clear area of the array or negative control spots as a background value.
• Compare corresponding signals on different arrays to determine the relative change in analyte levels between samples

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