Mcp 1

Equal amounts (30 μg) of protein extracts from brain of the animals (n = 6, for each group) were loaded and separated by SDS-PAGE using 7% or 12% acrylamide gradients. The membranes were incubated with monoclonal antibodies against NAPH oxidase (NOX)-I (1:1500, Sigma), NOX-II (1:500, Sigma), IL-1β (1:1000, proteintech), r-H2A (1:1000 Abcam), MCP-1 (1:1000, Millipore), TNF-⍺ (1: 1000, Cell Signaling) were used. Signals were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse, goat anti-rat or goat anti-rabbit IgG. Proteins were transferred to nitrocellulose membranes which were then incubated in the primary antibody solution overnight, followed by a washing procedure carried out three times within 15 minutes. The nitrocellulose membranes were then incubated with the second antibody solution for one hour at room temperature. The washing procedure was repeated three times within 15 minutes. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences), which was then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using Labwork software (UVP).

The plasma samples were prepared with ethylenediaminetetraacetic acid (EDTA) based on the CLIA standards. Milliplex Multiplex assays, commercially available customized plates, were used with few modifications to the manufacturer’s instructions. The following plasma biomarkers were purchased from EMD Millipore: total tau (catalogue number HND1MAG-39 K), phosphorylated tau (catalogue number HND1MAG-39 K), C-reactive protein (catalogue number HCVD3MAG-67 K), fibrinogen (catalogue number HCVD3MAG-67 K), plasminogen activator/inhibitor (catalogue number HADK1MAG-61 K), MCP-1 (catalogue number HCYTOMAG-60 K), eotaxin-1 (catalogue number HCYTOMAG-60 K), and YKL-40 (catalogue number HCMBMAG-22 K). The standards, controls, and subject samples were incubated with fluorescent-coded magnetic MagPlex-C microspheres overnight at 4 °C, and additionally incubated with a biotinylated detection antibody for 1 hour at room temperature (RT). The streptavidin–phycoerythrin conjugate was added and incubated at RT for 30 minutes, and then rinsed three times. The plates were read using Luminex xMAP technology (Bio-Rad Laboratories, Inc., Hercules, CA, USA) where microspheres were counted based on fluorescent reporter signals.

IMMP was synthesized by New England Peptide, Gardner, MA, USA. The commercial kits used in this experimental protocol were Glucose PAP, Triglycerides SDS and Cholesterol PAP (ELITech Group, Paris, France); Leptin, adiponectin and MCP-1 (Millipore, Missouri USA); Insulin (Crystal Chemic, USA); primary antibody and recombinant protein of IL-6, IL-10, IL4, TNF-α, IFN-γ and IL-1β (Peprotech Inc, NJ, USA); secondary antibody Anti-Rabbit IgG Peroxidase conjugated Affine Pure Goat (JacksonInmunoResearch, USA), and substrate 2,2´-Azino-bis(3-ethylbenzothiazoline 6-sulfonic acid) ABTS (Sigma-Aldrich, USA).

Plasma cytokine and chemokine concentrations were assayed in duplicate using a macrophage inflammatory protein 1α (MIP-1α) singleplex kit and a 22-analyte multiplex panel [granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma interferon (IFN-γ), interleukin-1 (IL-1) receptor agonist (IL-1RA), IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12/23(p40), IL-13, IL-15, IL-17, IL-18, monocyte chemoattractant protein 1 (MCP-1), MIP-1β, sCD40L, transforming growth factor α (TGF-α), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF)] (both from Millipore [catalog number PRCYTOMAG-40K]). The assays were performed according to the manufacturer’s recommended protocol, and the results were read using a FlexMAP three-dimensional array reader (Luminex Corp.). The data were analyzed using Bio-Plex Manager software (Bio-Rad).

Aliquots of plasma samples (stored at −80°C) were thawed and heat inactivated for 60 min at 55°C for removal from the BSL-3. Heat inactivated serum samples must be tested for residual live virus before removal from the BSL-3. Samples were then analyzed with Milliplex Non-Human Primate Magnetic Bead Panel containing the following analytes: TNFα, IL-6, IL-12/23p40, IL-8, MCP-1, IL1Ra, soluble CD40L, IL-15, IFNγ, IL-4 and IL-17 as per manufacturer's instructions (Millipore Corporation, Billerica, MA). Heat inactivation decreased the detection of the cytokines in this kit as follows: TNFα by 42%, IL-6 by 53%, IL-12/23p40 by 56%, IL-8 by 20%, MCP-1 by 33%, IL1Ra by 96%, IL-15 by 49%, IFNγ by 73%, and IL-17 by 19%. We were unable to determine the impact of heat inactivation on the levels of CD40L and IL-4 since the levels of these analytes were below detection in the test samples we subjected to this treatment.

Briefly, 3 × 10⁵ CD14⁺eWAT SVF macrophages were added to fibronectin-coated plates to assess adhesion and incubated for 4 hr at 37 °C before adhered cells were stained crystal violet and absorbance was quantified. Transwell migration assays were performed (Costar polycarbonate filters, 5-μm pore size) as previously described.^{79 (link)} Membranes and 12-well plates were coated with fibronectin (5 μl/mL; Life Technologies) overnight at 4 °C. CD14⁺eWAT SVF macrophages (0.3 × 10⁵ cells/well) were added to the upper chamber, and MCP-1 (100 ng/well; Sigma) in 0.8% agarose solution was added to the lower chamber to stimulate migration. Cells migrating into the lower chamber after 8 h of incubation were manually counted and presented as a percentage of cells migrated.