The cells were stimulated for 24 and 72 h with optimal doses of LPS (Sigma) and adiponectin (Peprotech, Inc., Cranbury, NJ, USA), respectively, in different variants: (1) LPS1000 ng/mL, (2) adiponectin 250 ng/mL, (3) adiponectin 1000 ng/mL, (4) adiponectin 250 ng/mL + LPS1000 ng/mL, and (5) adiponectin 1000 ng/mL + LPS1000 ng/mL. HFLS Basal Medium with BSA 0.5% (Sigma) was used as a negative control. The samples were performed in triplicate. After the time points, the cell pellets were used for total RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. LPS was derived from E. coli bacteria (lipopolysaccharides from Escherichia coli O55: B5) and was purchased from Sigma-Aldrich, St. Louis, MO, USA (Cat # L6529).
Adiponectin
Manufactured by Merck Group
Sourced in United States, Germany, Canada, Spain, Sao Tome and Principe, Switzerland
Adiponectin is a laboratory equipment product manufactured by Merck Group. It is a protein involved in the regulation of glucose and fat metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Leptin, by Merck Group (22 mentions)
Insulin, by Merck Group (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Resistin, by Merck Group (4 mentions)
Tnf α, by Merck Group (3 mentions)
Interleukin 6 (il 6), by Merck Group (3 mentions)
Adiponectin, by Thermo Fisher Scientific (3 mentions)
Insulin, by Mercodia (3 mentions)
Interleukin 6 (il 6), by R&D Systems (3 mentions)
Ghrelin, by Merck Group (3 mentions)
Leptin, by R&D Systems (3 mentions)
Anisomycin, by Merck Group (2 mentions)
Genistein, by Merck Group (2 mentions)
Tnf α and il 6, by Thermo Fisher Scientific (2 mentions)
Palmitic acid, by Merck Group (2 mentions)
Clotting activator tube, by Sarstedt (2 mentions)
Luminex multiplex kit, by Merck Group (2 mentions)
Serum insulin, by Crystal Chem (2 mentions)
Glycerol, by Merck Group (2 mentions)
Fgf21, by Merck Group (2 mentions)
84 protocols using adiponectin
1
Adiponectin and LPS Stimulation on HFLS Cells
2
Hormonal Regulation of Lymphocyte Function
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Lymphocytes were incubated with medium 199 (control) or with the following human hormones: adiponectin (Sigma, St. Louis, MO, USA) at 100 ng/mL, leptin (Thermo Fisher, Carlsbad, CA, USA) at 100 ng/mL, melatonin (Sigma, St. Louis, MO, USA) at 100 ng/mL, adiponectin + leptin at 100 ng/mL, and melatonin + leptin at 100 ng/mL. The stimuli were endotoxin-free. The hormone concentrations were determined according to previous research with mononuclear cells from human colostrum taken from eutrophic and obese puerperal women [6 (link),7 (link)].
3
Adipokine Effects on MCF10A Cells
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MCF10A cells were purchased from ATCC (ATCC® CRL-10317TM) and maintained in DMEM/F12 supplemented as described by Brugge lab1 and elsewhere. The cells were subcultured before reaching 80% confluence. For adipokine treatments the MCF10A cells were serum-starved overnight (Yan et al., 2012 (link)). Leptin (#L4146) and adiponectin (#SRP4901) were purchased from Sigma-Aldrich and added to final concentrations of 50–100 ng/ml (Leptin) and 25–250 ng/ml (adiponectin). The cells were incubated with adipokines for duration 24–72 h. As mechanosensitive Ca2+ channel inhibitor was used GSMTx4 (#ab141871, Abcam) in concentration 2.5 μM for 16 h.
For siRNA silencing, 25 nM ON-TARGET plus SMARTpoolTM siRNA library AMPK(#J-005027-06 Dharmacon, GE Healthcare) or Ob-R (Leptin receptor, #L-008015-00-0005 Dharmacon, GE Healthcare) were transfected into cells on 35 mm plates by using RiboJuice transfection reagent (#71115, Novagen) according to the manufacturer’s instructions. Cells were incubated for 72–96 h for efficient depletion of the target protein.
For siRNA silencing, 25 nM ON-TARGET plus SMARTpoolTM siRNA library AMPK(#J-005027-06 Dharmacon, GE Healthcare) or Ob-R (Leptin receptor, #L-008015-00-0005 Dharmacon, GE Healthcare) were transfected into cells on 35 mm plates by using RiboJuice transfection reagent (#71115, Novagen) according to the manufacturer’s instructions. Cells were incubated for 72–96 h for efficient depletion of the target protein.
4
Adiponectin signaling in blastocysts
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To study the effects of adiponectin on the expression of adiponectin signalling molecules [APPL1, AMPK, ACC, p38 MAPK, lipogenic molecules (PPARα, CPT1, cluster of differentiation 36 (CD36), fatty acid transport protein 4 (FATP4), fatty acid binding protein 4 (FABP4), HSL and lipoprotein lipase (LPL)) and the insulin/IGF-system (IGF1, IGF2, IGF receptor (IGF1R) and insulin receptor (InsR))], day 6 blastocysts were cultured in groups of three to five at 37°C in a water saturated atmosphere of 5% O2, 5% CO2, 90% N2 for one or 4 h with 1 μg/ml adiponectin (Biovendor, Germany) for RNA analysis. Control embryos were cultured without adiponectin. To prove AMPK-dependency, blastocysts were pre-cultured with Compound C, a specific AMPK inhibitor (10 μM, Sigma Germany) dissolved in dimethylsulfoxid (DMSO) for 30 min and subsequently cultured with 1 μg/ml adiponectin for 1 h. A DMSO group was included as solvent control for Compound C. For protein analysis, blastocysts were cultured with 1 μg/ml adiponectin for 15 min or 4 h in groups of six to nine. Incubation periods of adiponectin for protein and RNA analysis are based on our pervious study (Fischer et al., 2010 (link)) and the literature (Yoon et al., 2006 (link)). After culture, blastocysts were processed as describe above and stored at −80°C.
5
Hormone and Metabolite Profiling Protocol
6
Serum Biomarkers in Murine EAE
7
Comprehensive Biomarker Quantification Protocol
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Serum glucose, uric acid, C-reactive protein (CRP), LDL, and HDL cholesterol were quantified using conventional methods with an Olympus AU5400 automated clinical chemistry analyzer (Beckman-Coulter, Izasa, Porto, Portugal). Homocysteine was measured by chemiluminescent immunoassay using an Architect i2000 SR automated analyzer (Abbott).
Serum folate was quantified by time-resolved fluoroimmunoassay using an AutoDelfia automated analyzer (PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland).
Plasma insulin, leptin, and adiponectin were quantified by ELISA using commercially available kits according to the manufacturer's instructions (insulin and leptin: Millipore, St Charles, MO, USA; adiponectin: Invitrogen Corporation). According to the instructions, if the difference between duplicate results of a sample was over O15% (of the coefficient of variation), the sample was repeated whenever it was possible. Sensitivities of the assays were 0.2 ng/ml (insulin), 0.04 ng/ml (leptin), and 50 pg/ml (adiponectin).
Serum interleukin 1 beta (IL1b), interleukin 10 (IL10), and transforming growth factor-beta 1 (TGFb1) were measured by Luminex assay using the custom Milliplex Rat kits (Merck Millipore, Madrid, Spain), according to the manufacturer's protocols, by Luminex xMAP Multiplexing Technology platform.
Serum folate was quantified by time-resolved fluoroimmunoassay using an AutoDelfia automated analyzer (PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland).
Plasma insulin, leptin, and adiponectin were quantified by ELISA using commercially available kits according to the manufacturer's instructions (insulin and leptin: Millipore, St Charles, MO, USA; adiponectin: Invitrogen Corporation). According to the instructions, if the difference between duplicate results of a sample was over O15% (of the coefficient of variation), the sample was repeated whenever it was possible. Sensitivities of the assays were 0.2 ng/ml (insulin), 0.04 ng/ml (leptin), and 50 pg/ml (adiponectin).
Serum interleukin 1 beta (IL1b), interleukin 10 (IL10), and transforming growth factor-beta 1 (TGFb1) were measured by Luminex assay using the custom Milliplex Rat kits (Merck Millipore, Madrid, Spain), according to the manufacturer's protocols, by Luminex xMAP Multiplexing Technology platform.
8
Adipokine Measurements in SWAN Cohort
9
Protein Abundance Detection Protocol
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Protein extractions were performed as previously described44 (link),49 . Primary antibodies HAS2 (Santa Cruz Biotechnology, sc-34068) (1:200 dilution), HSL (Santa Cruz Biotechnology, sc-74489) (1:200 dilution), α Tubulin (Santa Cruz Biotechnology, sc-53030) (1:200 dilution), Actin (Sigma #A4700) (1:1000 dilution), Adiponectin (homemade) (1:1000 dilution) were used. Protein abundance was detected using the one of the following secondary antibodies: Goat anti-Mouse IRDye 680RD (Li-cor 926-68070), Goat anti-rabbit IRDye 800CW (Li-cor 925-32211) or Goat anti-Rat DyLight 800 (Thermo Fisher SA5-10024) at 1:10,000 dilutions. Antibody decorated membranes were then visualized on a Li-Cor Odyssey infrared scanner (Li-Cor Bioscience). The scanned data were analyzed using Odyssey Version 3.0 software (Li-Cor Bioscience).
10
Antibody Acquisition and Reagent Preparation
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Antibodies against p-AMPKα (T172) and pSAPK/JNK were purchased from Cell Signaling Technology (Danvers, MA, USA); Antibodies against AMPKα1, AMPKα2, and LKB1, and recombinant LKB1 protein were purchased from EMD Millipore (Gibbstown, NJ, USA); Antibodies against MLK3 and GST, HRP-conjugated second antibodies and protein A/G agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Myc antibody was from Sigma-Aldrich (St Louise, MO, USA). 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR), Anisomycin, sorbitol, oligomycin, TNFα, IL6 and Adiponectin, glutathione agarose and glutathione were purchased from Sigma-Aldrich (St Louise, MO, USA).
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