Animals were killed 1 day (P9, n = 9; 3mo, n = 8) after HI for CCL2 assay. Animals were deeply anesthetized with isoflurane and perfused with cold phosphate‐buffered saline (pH 7.4) (Life technologies). Brains were removed and hemispheres were separated. Hippocampi were dissected from the right hemisphere and placed in a precooled 2.0 mL Eppendorf tube in dry ice. Ice‐cold extraction buffer consisting of 50 mM Tris HCl (pH 7.3), 100 mM NaCl, 5 mM EDTA, 1 mM EGTA (all from Sigma‐Aldrich), and protease inhibitor cocktail (Completemini; Roche) was added, and the tissue was homogenized by sonication. Homogenates were centrifuged at 10,000g at 4°C for 10 min and the supernatants were collected. Protein concentration was determined using the BCA protein assay kit (Life technologies), and samples were aliquoted and stored at −20°C. CCL2 concentrations were determined using Quantikine Elisa kit (R&D systems). Samples were assayed in duplicates, and the assay was run according to the manufacturer instructions. Results were expressed as picogram/milligram protein (pg/mg protein).
Phosphate buffered saline ph 7.4
Manufactured by Thermo Fisher Scientific
Sourced in United States
Phosphate-buffered saline (pH 7.4) is a commonly used buffer solution that maintains a stable pH of 7.4. It is composed of a combination of sodium phosphate and sodium chloride in purified water. This solution is a fundamental tool in various laboratory applications, providing a physiologically relevant environment for a wide range of biological experiments and procedures.
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Lab products found in correlation
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Quantikine elisa kit, by R&D Systems (1 mentions)
Hydrochloric acid (hcl), by Thermo Fisher Scientific (1 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (1 mentions)
Optima lc ms grade, by Thermo Fisher Scientific (1 mentions)
Trehalose dihydrate, by Merck Group (1 mentions)
Igg alex fluor 647, by Thermo Fisher Scientific (1 mentions)
Amplex ultrared reagent, by Thermo Fisher Scientific (1 mentions)
Lipase b acrylic resin, by Merck Group (1 mentions)
α chymotrypsin from bovine pancreas, by Merck Group (1 mentions)
D glucose anhydrous, by Merck Group (1 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
N succinyl ala ala pro phe p nitroanilide, by Merck Group (1 mentions)
3 3 5 5 tetramethylbenzidine tmb liquid substrate system for elisa, by Merck Group (1 mentions)
Polyethylene glycol, by Carl Roth (1 mentions)
Poly vinyl alcohol, by Carl Roth (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
8 protocols using phosphate buffered saline ph 7.4
1
Hippocampal CCL2 Quantification Protocol
2
Synthesis and Characterization of (1E7-03) Analogs
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1E7-03 and its five analogs (purity above 98%) were synthesized by Enamine (Kiev, Ukraine) as previously described (Ammosova et al., 2014 (link)). Acetonitrile and water containing 0.1% formic acid (FA) were Optima LC/MS grade (Fisher Scientific, Fair Lawn, NJ, United States). High-purity nitrogen (99.9%) was purchased from Roberts Oxygen Co, Inc. (Rockville, MD, United States). Other reagents were of analytical grade. Dimethyl sulphoxide (DMSO), acetone, hydrochloric acid and sodium hydroxide were from Fisher Scientific (Fair Lawn, NJ, United States). Sodium acetate (pH 5.2) was from Quality Biological (Gaithersburg, MD, United States). Phosphate buffered saline (pH 7.4) was from Life Technologies (Grand Island, NY, United States).
3
Live Tissue Staining in Drosophila
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Wildtype OregonR Drosophila melanogaster were raised on standard cornmeal–yeast–agar medium at 25 °C and used for all experiments. For staining of living Drosophila melanogaster tissues, wandering third instar larvae were dissected in 1× PBS (Phosphate Buffered Saline) pH 7.4 (Life Technologies, Carlsbad, California USA) and the inverted front half of the larva was incubated with probes of 1 μM in 1× PBS for 1 h at room temperature. After single washing step with 1× PBS, isolated tissues were mounted in 1× PBS under a coverslip and sealed with nontoxic duplicating silicone (picodent, Wipperfuerth, Germany) to prevent evaporation of the medium during imaging.
4
Enzymatic Activity Assays Development
5
Cryopreservation and Cryo-Sectioning of Tissue Samples
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Surgically obtained tissue samples were transported in a 0.9% sodium chloride solution and then deep-frozen at −80 °C in O.C.T. compound (Sakura Finetek, Staufen im Breisgau, Germany) 10.24% polyvinyl alcohol (Carl Roth, Karlsruhe, Germany), 4.26% polyethylene glycol (Carl Roth, Karlsruhe, Germany) and 85.5% non-reactive ingredients). The cryostat tissue was sliced via cryotome Leica SM3050S (Leica Biosystems, Wetzlar, Germany) in a 5 μm wide section and acetone-fixated (Carl Roth, Karlsruhe, Germany) on microscope slides for 10 s. After 10 min of air drying, the tissue slides were stored at −80 °C. For further preparation, the tissue slides were incubated in acetone solution at −20 °C for 10 min and afterwards rehydrated with PBS (phosphate-buffered saline, pH 7.4, Life Technologies, Carlsbad, Germany).
6
Analyzing Peripheral Blood Biomarkers and Brain Microglia After GCRsim Exposure
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Seven days after GCRsim exposure, upon arrival at UCSF, blood was collected via the tail vein puncture in awake animals. Using a restrainer, a small nick was made in the tail vein using a scalpel and ~50 μl of blood was removed using a pipette and placed in a tube containing EDTA (Sigma-Aldrich). Blood was used to measure acute changes in peripheral blood biomarkers, discussed below.
Animals were lethally overdosed using a mixture of ketamine (10 mg/ml) and xylazine (1 mg/ml). Once animals were completely anesthetized, the chest cavity was opened. Mice were perfused with 1× phosphate-buffered saline (PBS) (pH 7.4) (Gibco) until the liver was clear (∼1 to 2 min). Following PBS perfusion, the whole brain was quickly removed, and one hemisphere was immediately processed for microglia counts by flow cytometry. From the remaining hemisphere, brain regions were dissected (hippocampus), snap-frozen on dry ice, and stored at −80°C.
Animals were lethally overdosed using a mixture of ketamine (10 mg/ml) and xylazine (1 mg/ml). Once animals were completely anesthetized, the chest cavity was opened. Mice were perfused with 1× phosphate-buffered saline (PBS) (pH 7.4) (Gibco) until the liver was clear (∼1 to 2 min). Following PBS perfusion, the whole brain was quickly removed, and one hemisphere was immediately processed for microglia counts by flow cytometry. From the remaining hemisphere, brain regions were dissected (hippocampus), snap-frozen on dry ice, and stored at −80°C.
7
Murine and Human Norovirus Propagation
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Murine norovirus-1 (MNV) was provided by Dr. H. Virgin from Washington University (St. Louis, MO, USA). MNV was propagated in the RAW 264.7 cell line as previously described and viral stocks were titrated by plaque assays (Gonzalez-Hernandez et al. 2012 (link)). HuNoV-positive stool samples HuNoV GI.5 (CFIA-FVR-022) and GII.4 (CFIA-FVR-019) were provided by the British Columbia Center for Disease Control (BCCDC). The preparation of HuNoV from clarified 10% stool samples was adapted from Houde et al. (2006 (link)). Briefly, the stool samples were diluted in 1× phosphate-buffered saline (PBS), pH 7.4 (Gibco, Canada) to obtain a 10% suspension and were homogenized by vigorous agitation. Suspensions were clarified by centrifugation (20,000×g, 15 min, 4 °C) and kept frozen at − 80 °C.
8
S-Protein Loaded Chitosan Nanoparticles
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S-TMC NPs were prepared by the ionotropic gelation method as previously described [14 (link)]. Briefly, a sodium tripolyphosphate (TPP, 0.167 mg/mL) solution containing S-protein (0.3 mg/mL) and a TMC solution (1 mg/mL) in a HEPES buffer (pH 7.4) with 1% (v/v) Tween 80 was mixed under continuous stirring for 1 h at room temperature. The S-TMC NPs were separated from unbound S-glycoproteins by centrifugation at 10,000× g for 10 min. The pelleted NPs were resuspended in 1× Phosphate-buffered saline (PBS) pH 7.4 (Gibco, Amarillo, TX, USA) for further analysis. The supernatant was collected and used for estimating loading efficiency (LE) by a protein quantitation assay with a Micro BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). The percentage of LE was determined as previously described [14 (link)]. The physical properties of the NPs, including the mean particle size, the polydispersity index (PDI) and their zeta potential were determined by a zetasizer (Malvern Instruments Ltd., Malvern, UK).
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