Mouse inflammation antibody array c1

To decipher cytokine expression profiles that may discriminate between mice that develop chronic nociception from those who fully recover after sciatic nerve crush injury, we blindly performed cytokine protein arrays (Mouse Inflammation Antibody Array C1, RayBiotech catalog # AAM-INF-1) on whole sciatic nerve homogenates from age- and sex-matched Gdnf CKO and Gdnf WT mice on day 7 after unilateral crush injury, with uninjured nerves (Sham surgery) from the same mice serving as internal controls (54 (link)). Day 7 was chosen for the assay as we had demonstrated that early recovery from injury occurred at this time point which coincided with maximal sciatic nerve Gdnf expression. We pooled 4 sciatic nerves (2 females and 2 males) from each experimental group, extracted protein from homogenates as previously published (54 (link), 57 (link)), and processed protein arrays concurrently according to the vendor’s protocol. Arrays were imaged together using a Bio-Rad ChemiDoc™ XRS imaging system. Digital images analyzed by semi-quantitative 2-D spot densitometry to determine relative protein expression with the AlphaEaseFC software program (ProteinSimple, San Jose, CA).

Approximately 1.0 × 10⁷ cells/100 mm dish were seeded in a 6-well plate and treated as described above. After incubation at 37°C, serum-free medium was harvested from cells pre-treated with SIN (50 or 100 μg/mL) for 2 h, co-stimulated with LPS (1 μg/mL) for another 24 h. The serum-free medium was then centrifuged at 700 × g for 10 min to remove cell debris, and the supernatant was collected and stored at −80°C for cytokine array analysis. Cytokine array was performed using a Mouse Inflammation Antibody Array C1 (RayBiotech, Norcross, GA, USA, CODE: AAM-INF-1) capable of semi-quantitative detection of 40 mouse proteins, following the manufacture's protocol. ImageQuant LAS4000 Scanner (GE Healthcare Corporate) was used for the densitometry analysis of the array. The Scanner analysis tool for AAM-INF-1 (RayBiotech) was used to automatically analyze the antibody array data.

Mice were perfused with 10 ml of ice-cold PBS and whole brain was extracted. Hemibrains were added to glass beads and RIPA Lysis buffer with protease inhibitors and then underwent homogenization for 60 s. The homogenate was collected, then centrifuged at 12,000 × g for 10 min at 4°C to remove insoluble material. The protein concentration of each sample was determined using a bicinchoninic acid assay and then normalized. The cytokines were analyzed using a membrane-based cytokine array (Mouse Inflammation Antibody Array C1; Raybiotech) that analyzes 40 inflammatory cytokines. All materials used were from this kit. Briefly, the membranes were blocked with blocking buffer and then 1 ml of each sample was added to each membrane and left to incubate overnight at 4°C. The membranes were then washed 3× each with the wash buffers provided, followed by a 2-h incubation with biotinylated antibody cocktail at room temperature. The washes were repeated and then the membranes were incubated with HRP-Streptavidin for 2 h at room temperature. Following another wash set, membranes were incubated with detection buffer for 2 min, then imaged using chemiluminescence. Data were extracted using ImageJ to obtain spot signal densities from each image, then analyzed using the analysis workbook provided by Raybiotech.

Measurement of inflammation-related markers was performed using the mouse inflammation antibody array C1 according to the manufacturer’s protocol (RayBiotech, Peachtree Corners, GA, USA). The intensity of the proteins expressed was analyzed using the ImageJ software.

M-CSF macrophages were plated in 96-well plates at a density of 50,000 cells in 200 μl of complete cell culture medium. Splenocytes were plated in 24-well plates at a density of 2.5 × 10⁶ cells in 0.5 ml of complete cell culture medium. Cells were challenged with the indicated stimuli for 24 h and cell-free supernatants were then harvested and tested for TNF-α release using a commercial ELISA kit (eBioscience, San Diego, CA). The stimuli used were Pam₃CSK₄ (100 ng/ml), Ultrapure Escherichia coli LPS (100 ng/ml) (Invivogen, San Diego, CA), and inactivated yeasts of C. albicans (2 x 10⁶ yeasts/ml). Triplicate samples were analyzed in each assay.
TNF-α and IL-6 levels in the secretomes produced by HSPCs were measured using commercial ELISA kits (eBioscience, San Diego, CA). Moreover, levels of 40 cytokines were determined in the secretomes using a mouse cytokine array (RayBio Mouse inflammation antibody array C1) according to the manufacturer's instructions (RayBiotech, Norcross, GA).