Capsular polysaccharides were isolated as described, with some modifications [5 (link), 18 (link)]. Briefly, cells were pelleted from cultures at 3,450 x g for 15 min at 4°C. Five mg of glass beads (4 mm, Fisher) per g of cells were added and the mixture was gently shaken by vortex for 1 min. A volume of 50 ml of distilled water was added per g of disrupted cells and centrifuged at 8000 x g for 10 min at 4°C. The supernatant was recovered, clarified in a 0.22 μm filter unit (Millipore) and lyophilized. To separate the capsular arabinomannan (AM) from the rest of capsular polysaccharides, the capsule residue was resuspended in 4 ml of distilled water and subjected to a chloroform:methanol:water extraction (1:1:0.9). The upper phase was recovered and incubated in a rotavapor at 40°C overnight. Proteinase K (Sigma) was added at 10 mg/ml in a 50 mM Tris-HCl pH 7.5, 10 mM CaCl2 buffer and incubated overnight at 37°C. The deproteinated solution was dialyzed for 3 d at 4°C in distilled water, lyophilized and chromatographed on a column (90 cm x 1.8 cm) of Bio-Gel P-10 (Bio-Rad) using 0.1 M NaCl in 0.1% acetic acid. Collected fractions of 4 ml were assayed for carbohydrate content by the phenol-sulfuric acid assay. Pooled fractions were dialyzed in water and lyophilized. The concentration of protein was determined on each isolation step by Bradford.
Glass beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Glass beads are small, spherical particles made of glass material. They are commonly used in various laboratory applications as a tool for mixing, stirring, or agitating liquids or suspensions. Glass beads provide a physical surface for these processes without introducing additional chemical components into the sample.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Sensititre auto inoculator, by Thermo Fisher Scientific (2 mentions)
Superscript 2, by Thermo Fisher Scientific (2 mentions)
0.22 μm filter unit, by Merck Group (1 mentions)
Biogel p10, by Bio-Rad (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Tert butylbenzene, by Merck Group (1 mentions)
Nitrone spin trap phenyl n tert butylnitrone, by Merck Group (1 mentions)
2 2 6 6 tetramethylpiperidin 1 yl oxyl tempo, by Merck Group (1 mentions)
Oxoid, by Thermo Fisher Scientific (1 mentions)
Agpath id one step rt pcr reagent, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna virus kit, by Macherey-Nagel (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Precellys evolution homogenizer, by Bertin Technologies (1 mentions)
G4649, by Merck Group (1 mentions)
Glass bottom dishes, by MatTek (1 mentions)
Papain, by Thermo Fisher Scientific (1 mentions)
21 protocols using glass beads
1
Isolation and Purification of Capsular Polysaccharides
2
Comparative Diagnostic Accuracy of Sputum Tests
3
Activated Charcoal-Based EPR Spectroscopy
4
Yeast Viability Reduction Assay
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Following wiping, samples were incubated for 1 h at 25 °C in 2 mL DE with 100 µg/mL proteinase K (Fisher Bioreagents™, Fisher Scientific, Loughborough, UK) and 1 g of glass beads (Fisher Scientific, Loughborough, UK). After incubation, samples were vortexed for 2 min and then serially diluted, and 3 × 10 µL2 drops of each dilution was plated onto tryptone soya agar (TSA; Oxoid, Thermo Fisher Scientific, Newport, UK). Reduction in yeast viability, expressed as a log10 reduction, was calculated as the difference between the number of yeasts recovered from untreated (control) and treated samples.
5
Fetal and Maternal Tissue Harvesting
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At necropsy, fetal and maternal tissues were surgically removed. All necropsies were performed by a board-certified pathologist and two technicians. Hysterotomy was performed on the pregnant macaques under inhalation anesthesia, and the fetus, placenta, fetal membranes, umbilical cord, and amniotic fluid were collected for detailed tissue dissection post-fetal euthanasia with an overdose of sodium pentobarbital (≥120 mg/kg). Shortly after, the mother was euthanized with an overdose of sodium pentobarbital (≥120 mg/kg). Each tissue was grossly evaluated in situ, and then excised, with further dissection with separate forceps and razor blades for each tissue to minimize risks for cross-contamination. Tissues were collected snap-frozen in Dulbecco’s minimum essential medium (DMEM) and RNAlater and homogenized to a liquid state20 (link) with glass beads (Fisher Scientific) or a 5 mm steel ball (Qiagen). If tissue remained after snap freezing and RNAlater archival, tissues were preserved in 10% neutral-buffered formalin and routinely paraffin-embedded and processed with hematoxylin and eosin (H&E) or as noted below.
6
Stool Sample Collection for Microbiome Analysis
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Participants collected stool samples at the beginning and end of each intervention period into RNAlater for bacterial measures using a fecal collection tube with a scoop in the lid (Sarstedt, Numbrecht, Germany) containing 5 mL preservation solution and 8–10 glass beads (3 mm; Fisher, Waltham, MA, USA) [15 (link)]. They were instructed to collect 2 pea-sized aliquots of stool and immediately, at the time of defecation, place the stool into the collection tube and mix well by shaking. The samples were delivered to the laboratory within 24 h and stored at −80 °C.
7
RT-qPCR Detection of MERS-CoV
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A previously published RT‐qPCR protocol was used to detect MERS‐CoV genome (Vergara‐Alert et al., 2017 ). Briefly, lymphoid samples (cervical and mediastinal lymph nodes, tonsil and thymus) were placed in tubes containing 500 μl Dulbecco's modified Eagle medium (DMEM) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mmol/L glutamine and 2 mm glass beads (Fisher Scientific, USA), individually homogenized at 30 Hz for 2 min by using a TissueLyser II (QIAGEN, Hilden, Germany) and stored at −70°C until use. Viral RNA was extracted by using a NucleoSpin RNA virus kit (Macherey‐Nagel, Düren, Germany) following the manufacturer's recommendations. The extraction products were tested by RT‐qPCR, which was performed by using AgPath‐ID One‐Step RT‐PCR reagents (Applied Biosystems, Foster City, CA, USA). The amplification was conducted on a 7,500 Fast Real‐Time PCR System (Applied Biosystems, USA) programmed under the following conditions: 50°C for 5 min, 95°C for 20 s and 45 cycles at 95°C for 3 s and 60°C for 30 s. Samples with a cycle threshold <40 were considered positive for MERS‐CoV RNA (Vergara‐Alert et al., 2017 ).
8
Genomic DNA Extraction from Bulk Leaf Samples
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For linkage map analysis, 10 young leaves were bulk harvested for each of the 87 F4:5 lines of POP1 and 59 F4:5 lines of POP2. For genotyping an additional 26 lines of POP1 and 29 lines of POP2, 6 young leaves of each line were bulk harvested at F4:7 generation. Genomic DNA of each line was then extracted using a modified CTAB method (Wen et al. 2014 (link)). Briefly, the bulked leaf tissue was first frozen at -80° for at least 24 hr, freeze-dried, ground with glass beads (Fisher Scientific, Pittsburgh, PA), and DNA concentration of each sample was measured with an ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA).
9
Quantification of Fecal IgA and Calprotectin
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For IgA quantification, frozen fecal samples were thawed and 0.2 g aliquots of feces were diluted in 2 mL PBS. After homogenization with 0.10 to 0.25 mm diameter glass beads (Fisher Scientific) using a Precellys Evolution homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) for 40 s at 6400 rpm, suspensions were centrifuged for 5 min at 13,000× g at 4 °C. Supernatants were collected and stored frozen at −80 °C prior to ELISA analyses following the manufacturer’s instructions (Canine IgA, FineTest, Wuhan, China). For calprotectin quantification, fecal extracts were prepared as previously described [36 (link)]. Briefly, frozen fecal samples were thawed and 1.3 g aliquots of feces were diluted 1:5 in fecal extraction buffer (20 mM CH3CO2Na and 3 mM CaCl2 [pH 7.6]) containing a protease inhibitor cocktail (1 tablet/25 mL, Roche, Basel, Switzerland). After homogenization by vigorous shaking for 30 min at room temperature, the suspensions were centrifuged for 20 min at 2100× g at 4 °C. The supernatants were collected and centrifuged for 30 min at 10,600× g at 4 °C. The final supernatants (fecal extracts) were stored frozen at −80 °C prior to ELISA analyses following the manufacturer’s instructions (Canine Calprotectin, MyBiosource, San Diego, CA, USA).
10
Phenolic Extraction and Quantification in Potatoes
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The method of extraction was adapted from Shakya and Navarre (2006) . Phenolic compounds were extracted in four replicates taken from freeze dried raw and cooked potatoes. Freeze-dried powder (200 mg) was mixed with 1.5 mL of extraction buffer (50% MeOH, 2.5% metaphosphoric acid, 1 mM EDTA, chilled to 4 o C) and 500 mg of glass beads (1.0 mm in diameter; Fisher Scientific). Tubes were shaken with a vortex for 10 min at room temperature and then sonicated at 10 °C for 10 min. After sonication, tubes were shaken again with a vortex for 10 min. Tubes were centrifuged at 2500 g at 4 o C for 10 minutes and the supernatant was transferred to a clean tube. Extractions were repeated three times and supernatants combined.
Samples were kept chilled at all times and not exposed to light. TPC of potato was measured by Folin-Ciocalteau method (Singleton and Rossi, 1965) . Potato extract (50µL) was mixed with Folin-Ciocalteau reagent (50 µL, 1 N), sodium carbonate (150 µL, 20% w/v) and distilled water (750 µL). After shaking by vortex, the mixture was incubated in the dark at room temperature for 45 min. Absorbance was measured at 765 nm using a spectrophotometer (Cecil, CE 7200 Double Beam UV/VIS Spectrophotometer). Different concentrations of 5-CQA (10-300 µg mL -1 ) were used to generate a standard curve.
Samples were kept chilled at all times and not exposed to light. TPC of potato was measured by Folin-Ciocalteau method (Singleton and Rossi, 1965) . Potato extract (50µL) was mixed with Folin-Ciocalteau reagent (50 µL, 1 N), sodium carbonate (150 µL, 20% w/v) and distilled water (750 µL). After shaking by vortex, the mixture was incubated in the dark at room temperature for 45 min. Absorbance was measured at 765 nm using a spectrophotometer (Cecil, CE 7200 Double Beam UV/VIS Spectrophotometer). Different concentrations of 5-CQA (10-300 µg mL -1 ) were used to generate a standard curve.
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