First, 500 μl of sucrose solution was removed from the top of the gradient without disturbing gradient composition. The cytosolic lysate (∼500 μl) was then layered on the surface of the gradient. To precisely balance the samples for ultracentrifugation we used 1× hypotonic lysis buffer when needed. The samples were centrifuged at 209 815 RCF for 2 h at 4°C in a SW 41 Ti rotor and a Beckman Coulter Ultracentrifuge Optima L-90K. Samples were eluted using either the gradient station (BioComp) or the Biologic LP pump (Bio-Rad) coupled to a Model EM-1 Econo UV detector (BioRad). Fractions (∼500 μl) were collected with either a Piston Gradient Fractionator (BioComp) coupled with a fraction collector (Gilson) or a model 2110 fraction collector (Bio-Rad). The precise location of the fractions along the UV-tracing was monitored using either the gradient profiler v1.25 (BioComp) software or the LP Data View v1.03 (Bio-Rad). TRI-reagent® was immediately added to each fraction and fractions were kept on ice prior to storage at −80°C.
Model 2110 fraction collector
Manufactured by Bio-Rad
Sourced in United States
The Model 2110 fraction collector from Bio-Rad is a programmable device used to automatically collect liquid samples into individual tubes or wells. It is designed to accurately dispense and distribute liquid fractions based on time or volume.
Automatically generated - may contain errors
Lab products found in correlation
Sephacryl s 300 high resolution, by GE Healthcare (2 mentions)
Glass econo column chromatography column, by Bio-Rad (2 mentions)
Amicon ultra centrifugal filter, by Merck Group (2 mentions)
Ultracel 10 kda ultrafiltration discs, by Merck Group (2 mentions)
Sephadex g 15 gel, by GE Healthcare (2 mentions)
Piston gradient fractionator, by BioComp Instruments (1 mentions)
Gradient station, by BioComp Instruments (1 mentions)
Optima l 90k ultracentrifuge, by Beckman Coulter (1 mentions)
Model em 1 econo uv detector, by Bio-Rad (1 mentions)
Gradient profiler v1, by BioComp Instruments (1 mentions)
Fraction collector, by Gilson (1 mentions)
Nuclepore polycarbonate membrane, by Cytiva (1 mentions)
Econo gradient pump, by Bio-Rad (1 mentions)
Flow adaptor, by Bio-Rad (1 mentions)
Glass econocolumns, by Bio-Rad (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Chromolith c18, by Merck Group (1 mentions)
Vacutainer, by BD (1 mentions)
Glucagon, by Peptide Institute (1 mentions)
Glp 1, by Peptide Institute (1 mentions)
10 protocols using model 2110 fraction collector
1
Subcellular Fractionation and RNA Isolation
2
Continuous Flow Experiments with Compartmentalized PEBs
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Flow experiments
were conducted similarly
to the description in previous work,33 (link) but
replacing the inflow of the desired enzymes with the desired volume
of PEBs, which remained compartmentalized in a Continuously Stirred
Tank Reactor (CSTR) during the experiment. The openings of the reactors
were sealed with Whatman Nuclepore polycarbonate membranes (5 μm
pore size) to prevent outflow of PEBs. Cetoni Low-Pressure High-Precision
Syringe Pumps neMESYS 290N were used to control the dispense of the
different solutions, prepared in Gastight Hamilton syringes (2500–10 000
μL), into the CSTR. The precise flow profile of the desired
flow rates was programmed using the Cetoni neMESYS software.
To detect and determine outflow concentrations from the CSTR, both
online and offline detection was employed. Online absorbance detection
was achieved with an Avantes AvaSpec2048 Fiber Optic spectrometer
and Avantes AvaLight 355 nm LED combined with a custom designed flow
cuvette provided to us by LabM8. Alternatively, offline measurement
could be achieved by means of connecting the outflow to a BioRad Model
2110 fraction collector. These fractions could subsequently be probed
for NADH absorbance using a Tecan Spark M10 platereader, or probed
for ATP, ADP, NAD+, and NADH using a Shidmadzu Nexera X3 HPLC.
Further details on the instrumentation and experimental protocols
can be found in theSupporting Information .
were conducted similarly
to the description in previous work,33 (link) but
replacing the inflow of the desired enzymes with the desired volume
of PEBs, which remained compartmentalized in a Continuously Stirred
Tank Reactor (CSTR) during the experiment. The openings of the reactors
were sealed with Whatman Nuclepore polycarbonate membranes (5 μm
pore size) to prevent outflow of PEBs. Cetoni Low-Pressure High-Precision
Syringe Pumps neMESYS 290N were used to control the dispense of the
different solutions, prepared in Gastight Hamilton syringes (2500–10 000
μL), into the CSTR. The precise flow profile of the desired
flow rates was programmed using the Cetoni neMESYS software.
To detect and determine outflow concentrations from the CSTR, both
online and offline detection was employed. Online absorbance detection
was achieved with an Avantes AvaSpec2048 Fiber Optic spectrometer
and Avantes AvaLight 355 nm LED combined with a custom designed flow
cuvette provided to us by LabM8. Alternatively, offline measurement
could be achieved by means of connecting the outflow to a BioRad Model
2110 fraction collector. These fractions could subsequently be probed
for NADH absorbance using a Tecan Spark M10 platereader, or probed
for ATP, ADP, NAD+, and NADH using a Shidmadzu Nexera X3 HPLC.
Further details on the instrumentation and experimental protocols
can be found in the
3
Exosome Isolation by Size Exclusion Chromatography
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The samples were centrifuged at 300× g for 10 min, followed by centrifugation at 3000× g for 10 min. The clarified supernatant was then concentrated to approximately 500 μL on a 100 KD Amicon Ultra Centrifugal filter (Millipore, Temecula, CA, USA). The exosomes were then isolated from the concentrated supernatant by size exclusion chromatography (SEC). Sephacryl S-300 High Resolution (GE Healthcare, Chicago, IL, USA) was packed on a glass econo-column chromatography column (BioRad, Hercules, CA, USA) (10 cm height, 1.5 cm diameter). The column was washed with 0.32% Sodium Citrate in PBS, and the supernatant was loaded onto the column and allowed to enter the resin by gravity flow. The eluate was collected in 23 fractions of 15 drops (~500 μL) on a Model 2110 Fraction Collector (BioRad, Hercules, CA, USA). For each fraction, the presence of exosomes was determined by nanoparticle tracking analysis as described below. The exosome-containing fractions were then further concentrated to 1/100th of the original supernatant.
4
FPLC Fractionation of Toluene Synthase
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FPLC fractionation was performed in an anaerobic globe box. Crude cell-free extract supernatants were applied to a Bio-Scale Mini CHT-II ceramic hydroxyapatite column (5-mL bed volume, 40-μm particle diameter; Bio-Rad, Hercules, CA, USA) with a Bio-Rad Econo Gradient Pump. The chromatographic conditions were as follows: using a flow rate of 1 mL/min and a binary eluent system of 10 mM and 500 mM potassium phosphate buffer (pH 7.5; also containing 2 mM DTT), the column was held at an initial phosphate concentration of 10 mM for 1 column volume and protein was then eluted with a linear gradient from 10 to 500 mM phosphate at a rate of 49 mM/mL. One-mL fractions were collected with a model 2110 fraction collector (Bio-Rad) and assayed for phenylacetate decarboxylase activity (as described above). Attempts were made to further purify toluene synthase activity with a quaternary ammonium anion exchange column (Bio-Scale Mini UNOsphere Q) after buffer exchange of active fractions from the hydroxyapatite column, however, these attempts were unsuccessful and are not further reported here.
5
Isolation of NK Cell Exosomes
6
Isolation and Purification of β2,6 FOS
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Two grams of E. herbicola levan (Sigma-Aldrich) were dissolved in 100 ml phosphate-buffered saline (PBS) (Oxoid) by autoclaving. The solution was cooled to room temperature before the addition of 100 nM Bt1760 GH32 endo-acting levanase and incubated at 37 °C for 30 min to partially digest the levan to a mixture of different size β2,6 FOS. The enzyme was then heat inactivated by boiling for 20 min. The resultant sample was freeze dried using a Christ Alpha 1-2 Freeze Drier at −45 °C. The freeze-dried FOS mixture was resuspended in 5 ml 50 mM acetic acid and loaded onto a column (two 2.5 × 80 cm2 Glass Econo-Columns, connected in series with a flow adaptor; Bio-Rad) packed with P2 Bio-gel size-exclusion resin (Bio-Rad) and pre-equilibrated with 50 mM acetic acid. The column was run at 0.25 ml/min using a peristaltic pump (LKB Bromma 2132 microperpex) and 2 ml fractions were collected continuously for 48 h using a Bio-Rad model 2110 fraction collector. Fractions were initially analysed by TLC and any that contained sugar were freeze dried to remove acetic acid.
7
Pharmacokinetics of [18F]T808 and [18F]T807
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The NMRI mice were anesthetized with isoflurane (2.5% in O2 at 1 L/min flow rate) and injected with [18F]T808 or [18F]T807 (∼3.7 MBq) via a lateral tail vein. They were killed by decapitation at 2, 10, 30, or 60 minutes PI (n = 3/time point). Blood was collected in EDTA-containing tubes (4 mL tubes; BD vacutainer, BD, Franklin Lakes, New Jersey) and stored on ice. Blood was subsequently centrifuged at 2330 ×g for 10 minutes to separate the plasma. Plasma (0.5 mL) was isolated and spiked with 10 µg of authentic T808 or T807 and analyzed by HPLC (Chromolith C18, 3.0 × 100 mm; Merck, Darmstadt, Germany) and eluted with gradient mixtures of 0.05 mol/L NaOAc pH 5.5 (A) and CH3CN (B; 0-4 minutes: isocratic 0% B, 0.5 mL/min; 4-14 minutes: linear gradient 0% B to 90% B, 1 mL/min; 14-17 minutes: isocratic 90% B, 1 mL/min). After passing through a UV detector at 254 nm coupled in series with a 3-in NaI(Tl) scintillation detector and connected to a single-channel analyzer, the HPLC eluate was collected as 1 mL fractions (model 2110 fraction collector; Biorad, Hercules, California). The radioactivity of fractions was counted in an automated γ-counter.
8
Labeling Peptides with Fluorescein
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Fifty μL of 10 -2 M fluorescein-5(6)-isothiocyanate (FITC, Sigma-Aldrich, MO, USA) dissolved in dimethylformamide was mixed with 50 μL of 10 -4 M GIP, GLP-1 or glucagon (Peptide Institute, Osaka, Japan) and, then 150 μL of Na 2 CO 3 /NaHCO 3 (0.1 M, pH 9.1) was added and continuously vortexed for 3 h in the dark at room temperature. The reaction mixture was then passed through a column prepacked with Sephadex G-25 Fine (GE Healthcare, Buckinghamshire, UK) saturated and eluted with 30 mM TES, 140 mM NaCl, 4 mM KCl (pH 7.4). Then the FITC labeled peptides were separated from unreacted FITC using the BioLogic TM LP System and Model 2110 Fraction Collector (Bio-Rad Laboratories, Hercules, CA, USA). Eluates were collected in fractions, each with a volume of 900 μL. The fraction with the highest fluorescence intensity determined at λex 485/ λem 528 was used for subsequent experiments.
9
Oligosaccharide Extraction from Maple Sap
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To remove the high-molecular-weight components, the maple sap was ultra-filtered using Ultracel 10 kDa ultrafiltration discs (Merck Millipore, Darmstadt, Germany). The filtrate then underwent gel filtration using Sephadex G-15 gel (GE Healthcare, Boston, MA, USA) on a column (1 m × 25 mm internal diameter; Bio-Rad Laboratories, Inc., Hercules, CA, USA) with water to obtain the oligosaccharide fraction. Each fraction, consisting of 200 drops, was collected using a Bio-Rad fraction collector Model 2110 (Bio-Rad Laboratories, Inc.).
10
Maple Sap Oligosaccharide Separation
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The excluding of the large-molecular-weight components in the maple sap was carried out by Ultracel 10 kDa ultrafiltration discs (Merck Millipore, Darmstadt, Germany). To separate the target oligosaccharide and other saccharides in maple sap, the filtrate was applied on an Econo-column (1 m × 25 mm internal diameter; Bio-Rad Laboratories, Inc., Hercules, CA, USA) packed with Sephadex G-15 gel (GE Healthcare, Boston, MA, USA). A Bio-Rad fraction collector Model 2110 (Bio-Rad Laboratories, Inc.) was used to collect each fraction, which consisted of 200 drops. Water was used for elution and washing.
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