Cytokine array panel a

Tumor tissues were collected from tumor-bearing mice 24 hours after the final treatment. The collected tissues were submerged in PBS and homogenized at 2500 rpm for 20 seconds using a Multi-Beads Shocker (Yasui Kikai Co. Osaka, Japan). After homogenization, Triton X-100 was added at a final concentration of 0.1%; protease inhibitors were also added. The samples were frozen at −80°C, thawed and centrifuged at 10,000 x g for 5 minutes to remove cell debris. Tissue lysates containing equal amounts of protein (400 μg) were subsequently used. A cytokine array was performed using cytokine array panel A (R&D Systems, Minneapolis, USA) according to the manufacturer's instructions. The results were analyzed using ImageQuant TL (GE Healthcare, Little Chalfont, UK).
For the CXCL2 ELISA assay, samples were prepared as described for the cytokine array. Mouse CXCL2 was assessed using an ELISA kit (DY452-05, R&D Systems, Minneapolis, USA) according to the manufacturer's instructions. The results were measured using a 96-well Mithras LB 940 Multimode Microplate Reader (Berthold Technologies GmbH & Co.KG, Bad Wildbad, Germany) at 540 nm.

Cytokine analysis was conducted using a mouse Cytokine Array Panel A (ARY006, R&D systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s protocol. Briefly, tumor and non-tumor parts of lung tissue homogenates were diluted and mixed with a cocktail of biotinylated detection antibodies. The sample/antibody mixture was then incubated with the Mouse Cytokine Array membrane containing 40 different anti-cytokine antibodies printed in duplicates. Any cytokine/detection antibody complex present was bound by its cognate immobilized capture antibody on the membrane. Following a wash to remove unbound material, Streptavidin-HRP and chemiluminescent detection reagents were added sequentially. The membrane was exposed to an X-ray film for 1–10 min. Light was produced at each spot in proportion to the amount of cytokine bound. The average signal (pixel density) of the pair of duplicate spots representing each cytokine was analyzed by ImageJ.

Cytokine Array Panel A (R&D Systems, #ARY006) was used to determine the cytokines present in the medium of co-cultured LSCs and fibroblasts. After 48 h of co-culture, the conditioned medium was transferred to the array, and the analysis was done according to the manufacturer’s instructions. The signals were detected and quantified using the Odyssey Infrared Imaging System (Li-Cor, Biosciences).

Cytokine levels in skin homogenates were measured with the mouse Cytokine Array Panel A (ARY006, R&D Systems, Bio-Techne). In brief, BALB/c mice were subjected to AFL or ID injection of Alum adjuvant (160 μg aluminum content) or left nontreated. Skin was dissected 24 hours later and homogenized in T-PER Reagent (78510, Thermo Fisher Scientific) followed by centrifugation at 18,000g for 10 minutes at 4°C. Supernatants were pooled for analysis of cytokine levels. In brief, membranes coated in duplicate with capturing antibodies against 40 cytokines were blocked and then incubated with tissue homogenates in the presence of a cocktail of biotinylated detection antibodies. After washing, membranes were incubated with HRP-conjugated antibodies (NA931, GE Healthcare Life Sciences, now Cytiva). After washing, membranes were incubated with SuperSignal West Femto Maximum Sensitivity Substrate (34095, Thermo Fisher Scientific) and then imaged under myECL Imager (Thermo Fisher Scientific).

The hemispheric tissue of the rat brains was homogenized in phosphate-buffered saline with protease inhibitor (Bertin technologies Montigny, Montigny-le-Bretonneux, France). Protein quantitation was conducted using the protocol included in the Pierce BCA protein assay kit. Proteome Profiler array was conducted using the Cytokine Array Panel A (R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions. Blots were visualized and evaluated by the ECLTM Western Blotting Analysis System (GE Healthcare, Chicago, IL, USA) and imaged using a LAS 4000 mini image analyzer (Fujifilm, Tokyo, Japan). The blots were quantized by HLImage++ (Western Vision software) to analyze this array.