Erlenmeyer flasks (125 mL) containing 20 mL 2 × SC−URA broth were inoculated with 1 × 106 cells/mL of the respective recombinant S. cerevisiae strain and grown aerobically on a rotary shaker at 200 rpm for 72 h at 30 °C. The cell-free supernatant (CFS) was harvested (1500 × g, 5 min) and filtered through 0.2 µm pore-size low-protein binding non-pyrogenic membranes (syringe filters, Pall Life Sciences, New York, USA). The antimicrobial activity of the CFS was determined using the agar well-diffusion assay [43 (link)] by spotting 100 µL of each sample in 6-mm wells that were cut into the surface of BHI 1% (w/v) agar seeded with a 1% (v/v) overnight culture of L. monocytogenes EDG-e. All plates were incubated at 37 °C for 18 h and examined for inhibition zones. In addition, the proteinaceous nature of the active supernatant was verified by treating the samples with 10 µg/mL trypsin (in 10 mM TRIS buffer, pH 8) for 1 h at 37 °C, and then determining activity against L. monocytogenes EDG-e as described above.
Syringe filter
Manufactured by Pall Corporation
Sourced in United States
Syringe filters are a type of lab equipment used to filter small volumes of liquid samples. They are designed to remove particulates and contaminants from the sample, ensuring the integrity of the analysis or experiment.
Automatically generated - may contain errors
Lab products found in correlation
Polyethylenimine (pei), by Polysciences (1 mentions)
Acetic acid, by Thermo Fisher Scientific (1 mentions)
Sodium phosphate dibasic, by Merck Group (1 mentions)
Potassium phosphate monobasic, by Merck Group (1 mentions)
Icap rq, by Thermo Fisher Scientific (1 mentions)
Hemoglobin assay kit, by Merck Group (1 mentions)
Deae sephadex a 25, by GE Healthcare (1 mentions)
Agilent 1200 hplc system, by Agilent Technologies (1 mentions)
Minimum essential medium (mem), by Merck Group (1 mentions)
Sunfire c18 analytical column, by Waters Corporation (1 mentions)
10 protocols using syringe filter
1
Antimicrobial Activity Screening of Recombinant Yeast
2
Producing HIV Pseudoparticles with Various Viral Spikes
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To produce HIV pps, HEK293 cells were transfected with pNL4.3-Luc R-E- in conjunction with plasmids encoding MERS-CoV S, SARS-CoV S, HCoV-229E S, MHV S, or VSV G using polyethylenimine (PEI, Polysciences Inc., Warrington, PA). Cell-free supernatants containing pps were collected at 48 h post-transfection, filtered through 0.45 µm syringe filters (Pall Life Sciences, Port Washington, NY), and stored at − 80 °C until use. All pseudoviruses were normalized for transducing activities by titration on HEK293 cells expressing appropriate CoV receptors, and were normalized for particle concentration by western blotting using HIV p24 antibodies.
3
HPLC Analysis of Myricetin and Drugs
4
Trace Element Quantification in Blood
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The samples of whole blood, plasma, and erythrocytes (n = 3 per group) were digested in 1.5 mL concentrated nitric acid (HNO3; 67%; Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 30 min and heated at 100 °C for 4 h with 1 mL concentrated hydrogen peroxide (H2O2; 30%; Sigma-Aldrich, St. Louis, MO, USA) as described in previous studies [30 (link)]. After diluting to a total volume of 10 mL with deionized water and passing through the syringe filters (0.22 μm; Pall Corporation, NY, USA), samples were subjected to inductively coupled plasma mass spectrometry with collision/reaction cell technology (ICP-CCT-MS; iCAP™ RQ; Thermo Fisher Scientific, Waltham, MA, USA) for total As and total Se detection. Standard solutions of As and Se were obtained from National Certified Reference Materials (Beijing, China) and used for standard curve graphing, and 2% (v/v) nitric acid was used as solution control.
To detect the protein-binding of As in erythrocytes (PB–As, PB-eAs), erythrocytes were hemolyzed with 0.5 mL of hypotonic Tris-NO3 (10 mM) on ice, and the concentrations of Hb in hemolysates were measured by Hemoglobin Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) for adjustment. Proteins in homogenates were precipitated by precooled acetone, and collected by centrifugation (14,000 rpm, 20 min). The protein pellets were then dissolved in Tris-NO3 (50 mM) for detection.
To detect the protein-binding of As in erythrocytes (PB–As, PB-eAs), erythrocytes were hemolyzed with 0.5 mL of hypotonic Tris-NO3 (10 mM) on ice, and the concentrations of Hb in hemolysates were measured by Hemoglobin Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) for adjustment. Proteins in homogenates were precipitated by precooled acetone, and collected by centrifugation (14,000 rpm, 20 min). The protein pellets were then dissolved in Tris-NO3 (50 mM) for detection.
5
Polymer Micelle Formation Protocol
6
Quantifying Glucosinolate Hydrolysis by LC-MS/MS
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Assays consisted of 50 µL crude beetle protein extracts or recombinant protein and 50 µL glucosinolate solution containing a mixture of 2-hydroxy-3-butenyl-, 3-butenyl-, 4-methylsulfinylbutyl-, 4-methylthiobutyl-, indol-3-ylmethyl-, benzyl-, 2-phenylethyl- glucosinolate, and sinalbin, each at 2 mM, in 20 mM MES buffer (pH 5.2). After incubation at 35 °C for 2 h, assays were stopped by adding 200 µL of pure methanol and 200 µL of 10% (w/v) aqueous DEAE-Sephadex A-25 (GE Healthcare Life Science, Pittsburgh, PA, USA) suspension to remove remaining glucosinolates. Samples were filtrated through a syringe filter (0.2 μm, Pall Corporation, Dreieich, Germany) into glass vials and stored at −20 °C until analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Assays performed with boiled protein samples served as a background control. Desulfo-glucosinolates were quantified by LC-MS/MS using an Agilent 1200 HPLC system (Agilent, Santa Clara, CA, USA) connected to an API3200 tandem mass spectrometer (AB Sciex Germany GmbH, Darmstadt, Germany) as described in Beran et al.15 (link). Multiple-reaction monitoring (MRM) was used to monitor analyte parent ion-to-product ion formation (Table S4 ). Data analysis was performed using Analyst Software 1.6 Build 3773 (AB Sciex). Desulfo-glucosinolates were quantified via external calibration curves.
7
Labeling and Tracking Transfused RBCs
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Donor KEL or wild type B6 RBCs were collected into anticoagulant preservative solution (CPDA, citrate phosphorus dextrose adenine, Jorgensen Labs, Henry Schein, Melville, NY), leukoreduced over a syringe filter (Pall Corporation, Port Washington, NY), and washed to remove residual citrate. Prior to transfusion in some experiments, B6 RBCs were labeled with chloromethylbenzamido 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (CM-DiI) and KEL RBCs were labeled with 3,3’-dihexadecyloxacarbocyanine perchlorate (DiO) according to the manufacturer’s instructions (Molecular Probes, Eugene, OR) and as previously described (5 (link)). Recipient mice were transfused via lateral tail with 50 µL of KEL RBCs (in addition to control wild type RBCs in RBC recovery experiments). Survival of the transfused RBCs was determined by calculating the ratio of circulating DiO to DiI RBCs in recipients at select time points post-transfusion.
8
Isolation and Culture of Primary Glial Cells
9
Mosquito Saliva Collection and Virus Amplification
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Saliva was collected from individual mosquitoes in groups of twenty at 2, 4, and 7 dpi using a forced salivation method54 (link)55 (link). A day before saliva collection, mosquitoes were deprived of sugar. Females were anesthetized by cold treatment and their wings and legs were removed. Proboscises of mosquitoes were inserted into 1 mm glass capillary tubes filled with 3–5 μl Cargille Type B immersion oil. After 1 h, saliva was collected from those capillaries in which droplets of saliva exuding from the proboscis were visible. Saliva samples were recovered from each capillary tube by centrifugation at 3,000 g for 15 min in a 1.5 ml Eppendorf tube containing 200 μl sample processing buffer. After addition of another 200 μl of sample processing buffer, each sample was vortexed and sterile-filtered using a 0.2 μm syringe filter (Pall Life Sciences, East Hills, NY, USA). To amplify virus from saliva samples, Vero cells seeded onto 24-well plates were inoculated with 180 μl saliva samples for 1 h at 37 °C before adding 1 ml of DMEM supplemented with 7% FBS. Cells were observed daily for the development of cytopathic effects (CPE) under an inverted microscope until 7 dpi. Virus titres from infected saliva samples were analysed by plaque assay as described above.
10
HPLC Analysis of Phenolic Compounds in ZO
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The HPLC profiling of the six major phenolic compounds in ZO was conducted using a Shimadzu Prominence LC-20A system (Kyoto, Japan) and LabSolution software (Version 5.53; SP3, Kyoto, Japan). The six analytes were separated using a SunFireTM C18 analytical column (4.6 × 250 mm, 5 μm; Waters, Milford, MA, USA) maintained at 40 °C. The mobile phase was composed of 0.1% (v/v) formic acid in distilled water (A) and 0.1% (v/v) formic acid in acetonitrile (B) under the following gradient elution conditions: 0–30 min, 30–70% B; 30–40 min, 70–100% B; 40–45 min, 100% B; 45–50 min, 100–30% B. The re-equilibration time was 10 min. The flow rate and injection volume were set to 1.0 mL/min and 10 μL, respectively. The standard stock solution of the six analytes was prepared at a concentration of 1.0 mg/mL with methanol and then diluted when used. The sample solution for quantitative analysis was ultrasonically extracted for 60 min by adding 10.0 mL of 70% methanol to 500.0 mg of lyophilized ZO. Finally, the prepared solutions were filtered through a 0.2 μm syringe filter (Pall Life Sciences, Ann Arbor, MI, USA) before injection into the HPLC instrument.
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