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  • Male C57Bl/6J (Stock #000664), GFP (C57Bl/6-Tg(UBC-GFP)30Scha/J; ubiquitous expression of Green Fluorescent Protein; Stock #004353) and TNFα -/- (B6.129S-Tnftm1Gkl/J; Stock #005540) mice were purchased from Jackson Laboratories at 8 weeks of age. Animals were housed 2 per cage in a pathogen-free environment on a 12 h light/dark cycle and were provided free access to food and water. Mice were euthanized by CO2 asphyxiation and processed immediately for spleen removal. All procedures in this study were approved by the Institutional Animal Care and Use Committee of the University of New Mexico. Splenocyte isolation was performed according to Kruisbeek [36] using wild type C57Bl/6J, GFP-expressing C57Bl/6J, or TNFα -/- mice. After spleens were excised, they were placed in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine, 100 µg/mL streptomycin sulfate and 100 units/mL penicillin (complete DMEM). Spleens were homogenized into single cell suspensions by gently disaggregating tissue between frosted ends of two microscopy slides, filtered through a 100 µm cell strainer, and then centrifuged at 800× g for 3 min at 4°C. Supernatants were discarded and cell pellets were resuspended in 1 mL ACK (Ammonium-Chloride-Potassium) Lysis Buffer (150 mM NH4Cl, 10 mM KHCO3 and 0.1 mM Na2-EDTA; pH 7.4) for 5-10 min to remove contaminating red blood cells. Complete DMEM was then added and cells were centrifuged again at 800× g for 3 min at 4°C. Supernatants were removed; cells were resuspended in complete DMEM and used for co-culture experiments after cell densities were determined by hemocytometer counting. Cell Culture and Adipocyte Differentiation: 3T3-L1 pre-adipocytes were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured and differentiated according to Gonzales and Orlando, 2008 [27]. Briefly, cells were grown in complete DMEM media at 37°C with 5% CO2 and passaged twice weekly. For differentiation, cells were seeded into 6-well cell culture plates coated with 1% gelatin. When cells reached confluency, they were incubated with 250 nM dexamethasone, 450 µM 3-Isobutyl-1-methylxanthine and 167 nM insulin for 3 days, then thoroughly rinsed with culture media and incubated for an additional 3–4 days with 167 nM insulin. Differentiation was confirmed by morphological changes, including intracellular lipid droplet accumulation, as confirmed by microscopic observation. 3T3-L1 cells were grown to confluency in 6-well culture plates and differentiated to mature adipocytes as described above. Adipocytes were then co-cultured with isolated splenocytes from wild type C57Bl/6J, GFP-expressing C57Bl/6J or TNFα -/- mice in direct and indirect contact systems in the presence or absence of LPS (E. coli 0111:B4 – 1 µg/mL; Sigma Aldrich, St. Louis, MO) in complete DMEM. For controls, cells were cultured individually with or without LPS, or cultured together in the absence of LPS. For indirect co-cultures, cells were cultured in a transwell system, with differentiated 3T3-L1 cells in the lower chamber and splenocytes (1.5×106 cells) seeded in a 0.4 µm hanging cell insert (Millipore, Billerica, MA). The hanging insert is constructed with a membrane having pores that are large enough to permit the passage of small molecules, yet small enough to prevent the passage of even the most motile of cell types. For direct co-cultures, splenocytes (1.5×106 cells) were added to differentiated 3T3-L1 cells, allowing direct contact between the two cell types. For TNFα recovery studies, co-cultures were supplemented with 0, 0.3 or 10 ng/mL purified murine TNFα (Cell Signaling, Danvers, MA). In NF-κB inhibitor experiments co-cultures were incubated with 2 µM Bay11-7082 (Calbiochem, La Jolla, CA). Murine TNFα, IL-6, IL-1β, MCP-1 and IL-10 levels in individual cultures and co-culture supernatants were measured by ELISA Ready-Set-Go kit (eBioscience, San Diego, CA) according to the manufacturer's directions. Media samples were diluted accordingly to ensure samples were within the detection kit sensitivity, specifically for MCP-1 and IL-6. After 24 h of direct co-culturing of 3T3-L1 cells and splenocytes from GFP mice, cells were sorted by fluorescence-activated cell sorting (FACS). Co-cultures were trypsinized and centrifuged at 800× g for 3 min at 4°C. Supernatants were removed and cells were resuspended in complete low serum (0.5% FBS) DMEM media with 5 mM EDTA and passed through a 100 µm cell strainer (BD Falcon) to ensure single-cell suspensions. GFP-positive and negative cells were sorted using the Beckman Coulter Legacy MoFlo high-speed sorter into separate tubes for subsequent mRNA extraction. RNA Isolation and Quantitative RT-PCR Analysis: Sorted 3T3-L1 cells and splenocytes were homogenized using the QIAshredder (Qiagen, Valencia, CA) and total RNA was isolated with RNeasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer's recommendations. RNA was converted into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Levels of F4/80, IL-6, adiponectin, MCP-1, TNFα and IL-10 were measured by quantitative Real-Time PCR (qRT-PCR), which was carried out using the LightCycler 480 SYBR Green I Master Mix chemistry (Roche Diagnostics, Indianapolis, IN) and analyzed on the LightCycler 480 instrument (Roche Diagnostics, Indianapolis, IN). Information on primer sequences, annealing temperatures, fragment sizes and genes can be found in Table 1. The reaction cycling parameters for IL-10 were performed as a 3-step qRT-PCR with a pre-incubation step at 95°C for 5 min and amplification for 45 cycles at 95° for 10 sec, 60°C for 15 sec and 72°C for 1 sec. All other genes were amplified in a 2-step qRT-PCR reaction with pre-incubation at 95°C for 15 min, followed by 40 cycles of amplification at 95°C for 15 sec and the annealing temperature (Table 1) for 1 min. A melting curve analysis was performed in each experiment for all genes to confirm specificity of single-target amplification. Gene expression changes were calculated using the relative standard curve method [37] and 36B4 mRNA levels were used as a normalizer. All samples were amplified in triplicate. Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0077306.t001 Gene and primer information for qRT-PCR. 36B4 annealing temperature varied according to target gene being analyzed. IL-6, interleukin-6; IL-10, interleukin-10; MCP-1, monocyte chemoattractant protein-1; TNFα, tumor necrosis factor alpha; and 36B4, 60S acidic ribosomal protein P0.
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