The peanut extract was prepared as previously described by Pi et al. (2015[22 (link)]). Raw peanuts were lightly blanched at 100 °C for 3 min and pulverized using a BioPulverizer (Biospec Products, Inc. Bartlesville, OK, USA). Pulverized peanuts were then mixed with phosphate buffered saline (Roche, Indianapolis, IN, USA) at a 1 kg:4 L ratio. A homogenizer (OMNI GLH) was used to further homogenize the peanut-PBS mixture. The mixture was stirred overnight at 4 °C, then centrifuged for 30 minutes at 100 x g at room temperature. The aqueous fraction was centrifuged for 30 more minutes at 100 x g at room temperature to remove residual lipids. The remaining aqueous fraction, or “peanut extract”, was collected and stored at -80 °C until use. A Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) was used to determine the protein concentration. The extract was stored so that each aliquot was thawed only once prior to use.
Phosphate buffered saline (pbs)
Manufactured by Roche
Sourced in Germany, United States, Switzerland
PBS (Phosphate Buffered Saline) is a commonly used buffer solution that maintains a physiological pH and osmolarity. It is a versatile laboratory reagent that serves as a diluent, washing, and stabilizing solution for various biological applications.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Biopulverizer, by Biospec (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Golgiplug, by BD (2 mentions)
Collagenase, by Roche (2 mentions)
Dispase, by Roche (2 mentions)
Monensin, by BioLegend (2 mentions)
40 μm cell strainer, by BD (2 mentions)
Dnase, by Roche (2 mentions)
O 1602, by Cayman Chemical (1 mentions)
Ml 193, by Cayman Chemical (1 mentions)
Lipopolysaccharide lps from salmonella typhimurium, by Merck Group (1 mentions)
Recombinant cytokines, by Thermo Fisher Scientific (1 mentions)
Amine coupling kit, by Cytiva (1 mentions)
Microplate reader, by Olympus (1 mentions)
Dimethyl sulfoxide (dmso), by Roche (1 mentions)
3 isobutyl 1 methylxanthine ibmx, by Merck Group (1 mentions)
Alexa 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
26 protocols using phosphate buffered saline (pbs)
1
Peanut Extract Preparation Protocol
2
Peanut Extract Immunogen Preparation
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A peanut extract was prepared in order to provide an immunogen that would elicit an IgE response upon injection. For this purpose, a peanut extract was prepared according to the method of deJonge et al. [9 (link)] with minor modifications: Raw peanuts were lightly blanched at 100°C for 3 min, pulverized using a BioPulverizer (Biospec Products, Inc. Bartlesville, OK, USA) and mixed with phosphate buffered saline (Roche, Indianapolis, IN, USA) at a 1kg:4L ratio. A homogenizer (OMNI GLH) was used to further homogenize the peanut-PBS mixture. The mixture was stirred overnight at 4°C, then centrifuged for 30 min at 100 x g at room temperature. The aqueous fraction was centrifuged for 30 more min at 100 x g at room temperature to remove the residual lipids. The remaining aqueous fraction, or “peanut extract”, was collected and stored at -80°C until use. The protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) according to manufacturer’s instructions. The extract was stored so that each aliquot was thawed only once prior to use.
3
Microglia Activation and Receptor Modulation
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KIT C and KIT H were synthesized as described previously [17 (link)] by the Karlsruher Institute for Technology (KIT), Institute of Organic Chemistry, dissolved in DMSO (Merck KGaA, Darmstadt, Germany) and used in final concentrations of 0.1–25 µM. The GPR55 agonist O-1602 and GPR55 antagonist ML 193 (both from Cayman Chemicals, Ann Arbor, MI, USA, distributed by BioMol, Hamburg, Germany) were dissolved in DMSO and used in final concentrations of 5 µM (O-1602) and 25 µM (ML 193). Human interleukin (IL)-1β (100,000 U/mL in phosphate buffered saline (PBS)) was purchased from Roche Diagnostics (Manheim, Germany) and was used at a final concentration of 10 U/mL for the experiments. 5 mg/mL lipopolysaccharide (LPS) from Salmonella typhimurium (Sigma-Aldrich GmbH, Taufkirchen, Germany) was dissolved in PBS as stock and diluted with distilled water for a final concentration of 10 ng/mL in primary microglia cultures.
Figure 9 shows the chemical structure of KIT C and KIT H that were already introduced in a previous paper [17 (link)], as well as the structures of O-1602 and ML 193 obtained from the manufacturer’s (Cayman Chemicals, Ann Arbor, MI, USA) website.
4
Permanent Coronary Ligations and Proliferation Markers
5
Dextran Binding Interaction Protocols
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Dextran (MW = 75 kDa)
was purchased from Thermo Fisher Scientific, dialysis membranes from
Spectrum, and recombinant cytokines from PeproTech. For ITC and SPR
samples, phosphate-buffered saline (PBS; Roche Diagnostics GmbH, Mannheim,
Germany); sterile dimethyl sulfoxide (DMSO) USP >99.9% (Stemsol,
Protide
Pharmaceuticals Inc., IL, USA); C1 carboxymethylated, matrix-free
chip series S (BR100535); and the Amine Coupling Kit (BR100050) were
obtained from Cytiva. Other solvents and general reagents were purchased
from TCI America or Sigma-Aldrich and used as received unless otherwise
indicated. 1H NMR spectra were acquired at 500 MHz (Varian
Unity Inova), and chemical shifts are reported relative to the residual
solvent peak.
was purchased from Thermo Fisher Scientific, dialysis membranes from
Spectrum, and recombinant cytokines from PeproTech. For ITC and SPR
samples, phosphate-buffered saline (PBS; Roche Diagnostics GmbH, Mannheim,
Germany); sterile dimethyl sulfoxide (DMSO) USP >99.9% (Stemsol,
Protide
Pharmaceuticals Inc., IL, USA); C1 carboxymethylated, matrix-free
chip series S (BR100535); and the Amine Coupling Kit (BR100050) were
obtained from Cytiva. Other solvents and general reagents were purchased
from TCI America or Sigma-Aldrich and used as received unless otherwise
indicated. 1H NMR spectra were acquired at 500 MHz (Varian
Unity Inova), and chemical shifts are reported relative to the residual
solvent peak.
6
MTT Assay for Cell Viability
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The cell viability was detected by the MTT assay. 1 × 104 THP-1 cells were inoculated in 96-well plates for 24 h and incubated with 20 μL MTT reagent which dissolved in phosphate buffer (PBS) (Roche, Basel, Switzerland, 5 mg/ml) at 37 °C for 4 h. The culture medium was removed and 150 μL dimethyl sulfoxide (DMSO, Roche, Basel, Switzerland) was added. A microplate reader (Olympus, Tokyo, Japan) was used to evaluate cell viability with absorbance of 490 nm.
7
Adipogenic Differentiation of SVF Cells
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SAT and VAT were dissected and then digested in digestion medium (collagenase D 1.5 unit/mL and dispase II 2.4 unit/mL in sterilized phosphate-buffered saline [PBS]; Roche, Mannheim, Germany) at 37℃ for approximately 30 minutes, which was stopped by adding complete medium (Dulbecco's modified Eagle's medium [GIBCO, ThermoFisher Scientific, Waltham, MA USA]/F12 containing 10% foetal bovine serum [FBS] and 100 µg/mL penicillin and 0.1 mg/mL streptomycin). After centrifugation, the cell suspension was filtered through a cell strainer (50 to 70 µm diameter) and centrifuged at 700×g for 10 minutes. The SVF cells were re-suspended in complete medium and plated on collagen-coated six-well plates. After reaching 95% to 97% confluence, the cells were initiated to differentiate via addition of induction medium (complete medium with addition of insulin [GIBCO] 5 µg/mL, indomethacin 125 µM, dexamethasone 2 µg/mL, and 3-isobutyl-1-methylxanthine [IBMX; Sigma-Aldrich, St. Louis, MO, USA]). After 48 hours of differentiation, the medium was changed to maintenance medium (complete medium with addition of insulin 5 µg/mL, GIBCO) for 24 hours to complete one differentiation cycle. The SVF cells underwent two adipogenic differentiation cycles.
8
Lcp1 Immunostaining in Zebrafish Larvae
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For Lcp1 immunostaining, larvae were anesthetized with 200 μg/ml tricaine and then immediately fixated in 4% paraformaldehyde in PBS (phosphate-buffered saline, pH 7.2) for 16 h at 4 °C. After fixation, the larvae were rinsed in PBS-DTx (phosphate-buffered saline with 0.5% DMSO and 0.3% Triton X-100) and treated with proteinase K (10 μg/ml in PBS-DTx; Roche) for 10 min at 37 °C. The larvae were blocked in 5% normal sheep serum (Sigma-Aldrich) in PBS-DTx for 2 h at room temperature, incubated with Lcp1/L-Plastin antibody (a gift from Dr. Anna Huttenlocher, University of Wisconsin, USA) in 1:1000 dilution at 4 °C overnight and subsequently incubated with Alexa-488 conjugated secondary antibody (1:200; Invitrogen) for 2 h at room temperature. The larvae were washed with PBS-DTx and stored at 4 °C until imaging.
9
Spermatozoa DNA Extraction Protocol
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Spermatozoa pellets of samples in the Method 1 group (Fig 1.1 ) were defrosted at room temperature and washed in PBS (Roche), followed by centrifugation at 400 x g for 10 min and supernatant removal. Cells were resuspended in lysis buffer (Tris-HCL 25 mM, pH 8.1, EDTA 5 mM, SDS 1%, proteinase K 0.4 mg/mL) and incubated at 55 °C for 6 h with gentle agitation. Lysates (Fig 1.2 ) were phenol-chloroform extracted and the DNA recovered by ethanol precipitation in the presence of ammonium acetate. DNA precipitates were resuspended in TE buffer (Tris-HCl 10mM, pH 8.1, EDTA 1mM). RNA remaining in the solution was digested with RNase A (20μg/mM) (Qiagen) at 37 °C for 20 min. Processed DNA samples were stored in TE buffer at -80 °C (Fig 1.3 ).
10
Isolation of Stromal Vascular Cells
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Stromal vascular cells (SVCs) were isolated from samples using a well-established collagenase-based method [13 (link), 14 (link)]. In brief, harvested epididymal fat pads were soaked in PBS (Gibco) and 2% BSA (Gibco), after which they were pulverized into pieces 1-2 mm in diameter. Next, collagenase (Sigma) and DNase I (Roche) were added to the samples, after which the reactions were shaken for 20 min at 37°C with the addition of 2% BSA/PBS and 5 mM EDTA. After filtration through 100 μM nylon mesh (BD Science), the mixtures were centrifuged at 1000 rpm for 3 min to remove the supernatants. The pellets at the bottom were mixed with PBS and 2% FBS (Sigma). The samples were filtered again using the 100 μm nylon mesh to remove unnecessary tissue. After centrifugation at 200 rpm for 10 min, SVCs were obtained from the bottom of the samples.
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