Human cytokine antibody array

To measure the release of cytokines, Human Cytokine Antibody Array from RayBiotech was used. Conditioned media were collected from cancer cells as summarised in Fig S3. Cytokine values from the unconditioned media used for conditioning were subtracted from the values obtained in the media from cancer cells. Consequently, cytokine levels in the media from cancer cells were subtracted from the values obtained in the media from fibroblasts. The kit was used according to the manufacturer’s instructions and analysed using supplied positive and negative controls for all the assays. We used the same control media for all conditions tested, and all the membranes were prepared at the same time. The values between different cell types were not compared, but the trend in cytokines released is presented on separate graphs for all conditions tested.
Citrate in the media from fibroblasts was measured using Citrate assay kit (Sigma-Aldrich) according to the manufacturer instructions.

Cytokine levels in control and VP-treated samples were determined using human cytokine antibody array (Ray Biotech, Cat. No. AAH-CYT-5-8) (Supplementary Table S3) as per manufacturer instructions. Using this array, we assayed the expression of 80 cytokines in OVCA cell lines. Briefly, the membranes from the cytokine array kit were incubated with control and VP (Fa0.5) treated cell lysates (500 μg of total protein) overnight at 4 °C (n=1). The membranes were then processed as per manufacturer and then assayed using chemiluminescence technique. Spots were identified and local background subtracted. By comparing the signal intensities, relative levels of cytokines were established.

440 cytokines were measured by a sandwich method based on the antibody array technique (Human Cytokine Antibody Array, RayBiotech, Norcross, GA, USA). Briefly, 100ul of serum was added to a chip containing 440 primary antibodies after 1:2 dilution of the serum sample and incubated at 4℃ overnight incubation. The chips were first washed 10 times with wash I diluted with deionized water for 10 s each shake, and then cleaned 6 times with wash II diluted with deionized water for 10 s each shake, then, biotinylated secondary antibodies were added and incubated for 2 h on a shaker at room temperature. After cleaning the chip again using the method described above, Cy3-conjugated streptavidin was added and incubated at room temperature in the dark for 1 h. InnoScan 300 Microarray scanner (Innopsys) is used to measure the fluorescent signal values. Then, using Mapix software will fluorescent the signal value into quantitative values.

The blood samples from both STS (n = 9) and saline control (n = 9) groups after 5‐day treatment were prepared and assessed the variable cytokines using a Quantibody Human Cytokine Antibody Array consisting of 440 different antibodies spotted in quadruplicate onto eight slide chips (RayBiotech, Norcross, GA, USA). The signal was acquired by fluorescence detection and quantified, and the relative expression levels of cytokines were determined by comparison between groups according to the manufacturer's instructions.
Gene ontology (GO) analysis in the set of differentially expressed factors was performed with DAVID Bioinformatics Resources 6.8 databases to investigate the associated biological process, cellular component and molecular function.13 Functional categories were enriched and the top 10 GO functional categories were selected. Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathway analysis to discover a relation that was not easily visible from the changes of individual proteins.

After completing the basic CSF analysis, such as cytological and biochemical examinations, the remaining CSF was centrifuged at 1,800 g at 4°C for 10 min, and the supernatant was aliquoted in 0.5-ml polypropylene tubes and stored at −80°C until further analysis.
The profiles of cytokines and inflammatory mediators were examined using a human cytokine antibody array (RayBiotech, USA) containing 80 cytokines and inflammatory mediators related to immune and inflammatory responses (Supplementary Table 1) according to the manufacturer’s instructions.

For cytokine array analysis, peripheral blood samples were collected from EOLP patients (n = 6) and NEOLP patients (n = 5), and age and sex matched to healthy controls (n = 5). The human cytokine antibody array (RayBiotech, Norcross, GA) capable of measuring 40 cytokines was used according to the manufacturer's instructions. Briefly, plasma was collected and stored at −80°C until use. While glass array slides were thawing to room temperature, the slides were blocked with 1× blocking buffer and incubated overnight at 4°C with 100 μL of plasma. Slides were washed and then incubated with 80 μL of biotin‐conjugated anticytokines at room temperature for 1‐2 hour. Slides were washed again and incubated at room temperature for 1 hour with Cy3 equivalent dye‐conjugated streptavidin. Each glass slide contained positive and negative internal controls. Fluorescence was detected with a GenePix 4000 scanner (RayBiotech, Sunnyvale, CA) using the Cy3 channel, and signal intensity was obtained with the scanning software. The RayBio Analysis Tool software (RayBiotech) was used to evaluate signal intensities after normalizing to the positive control and subtracting background noise. Changes in individual cytokines of ≥1.2‐fold increase and ≥0.8‐fold decrease were considered significant, as recommended by the manufacturer.