The PPLC gSEC column [1 ] (Fig 1 ) was packed with size-exclusion dextran-based Sephadex™ G-50 fine (17-0042-01), G-75 (17-0050-01), G-100 (17-0060-01) and 2% agarose-based gel filtration Sepharose CL-2B beads from Cytiva (formerly GE healthcare, Marlborough, MA, USA). The column was prepared by layering the beads from the smallest (G-50) to largest (Sepharose CL-2B) in a 25cm x 0.5cm Econo-Column® (Bio-Rad, Hercules, CA, USA) at room temperature by gravity. Analytes were eluted with 1x phosphate-buffered saline (PBS). Fractions were collected in Greiner UV-Star® 96-well plates using a FC204 fraction collector (Gilson, Middleton, WI, USA), with 12 drops per well. UV-Visible spectroscopy (absorbance) and fluorescence of the fractions were measured using a Synergy H1 plate reader. Fractions in each peak shown in the absorbance profiles (Fig 2 and Fig 3 ) were pooled and stored at −80°C. The BEV samples (peak 1) from each donor were thawed once to measure BEV ζ-potential, protein concentrations, EV marker western blots and transmission electron microscopy (TEM) images. They were thawed a second time for proteomic analysis. They were thawed a third time for proteomic validations by western blot. The samples were re-stored at −80°C after each thaw.
Fc204 fraction collector
Manufactured by Gilson
Sourced in United States
The FC204 fraction collector is a laboratory instrument designed to automatically collect liquid samples in a series of containers or tubes. It is capable of accurately dispensing pre-determined volumes of liquid into multiple fractions. The FC204 can be programmed to collect fractions at specified time intervals or based on other triggering events, making it a versatile tool for a variety of applications in analytical chemistry, biochemistry, and other scientific fields.
Automatically generated - may contain errors
Lab products found in correlation
Automated water bath, by Thermo Fisher Scientific (2 mentions)
Instat, by GraphPad (2 mentions)
Econo column, by Bio-Rad (2 mentions)
G50 fine, by GE Healthcare (1 mentions)
Sepharose cl 2b beads, by GE Healthcare (1 mentions)
Sepharose cl 2b, by Bio-Rad (1 mentions)
Franz diffusion cell, by PermeGear (1 mentions)
Gallic acid, by Carl Roth (1 mentions)
Hyperoside, by Carl Roth (1 mentions)
Isoquercitrin, by Carl Roth (1 mentions)
Polyamide, by Carl Roth (1 mentions)
Spd m10a vp pda detector, by Shimadzu (1 mentions)
Sunfire c18 column, by Waters Corporation (1 mentions)
Esquire 3000 plus iontrap mass spectrometer, by Bruker (1 mentions)
Agilent 1100 system, by Agilent Technologies (1 mentions)
Avance 3 500 mhz spectrometer, by Bruker (1 mentions)
Quercetin, by Merck Group (1 mentions)
Sepharose 4b resin, by Merck Group (1 mentions)
Poly prep chromatography column, by Bio-Rad (1 mentions)
Costar 3370, by Corning (1 mentions)
11 protocols using fc204 fraction collector
1
Fractionation and Characterization of Extracellular Vesicles
2
Preparation of Small Unilamellar Vesicles
3
In vitro Evaluation of Insulin-Loaded Microneedle Delivery
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The delivery of insulin loaded on the MNs was studied, using abdominal porcine skin. The in vitro testing was carried out in Franz diffusion cells (PermeGear, Inc., Hellertown, PA, USA). Dermatomed skin samples were placed in phosphate buffered saline (PBS; pH 7.4) for 1 hour prior to experimentation. The MN arrays were inserted into the skin samples using manual finger pressure. The skin and MN array was mounted onto the donor compartment of a Franz diffusion cell. The temperature of the Franz cells was maintained at 37 °C using an automated water bath (Thermo Fisher Scientific, Newington, USA). Sample fractions (6–6.5 mL/h) were collected using an auto-sampler (FC 204 fraction collector, Gilson, Middleton, WI, USA) attached to the Franz diffusion cells. Statistical analysis for the drug release was performed by using a Mann–Whitney nonparametric test (InStat, GraphPad Software Inc., San Diego, CA, USA), where samples are considered to be statistically significant at p < 0.05.
4
HPLC Purification and Characterization of Polyphenols
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Quercetin (12 , >98%) was purchased from Sigma-Aldrich. Gallic acid (1 , >98%), hyperoside (8 , >95%), isoquercitrin (10 , >95%), and polyamide (particle size: 0.05–0.16 mm) were from Carl Roth. HPLC-grade acetonitrile and methanol (Reuss Chemie AG), and distilled water were used for HPLC separations.
Preparative HPLC was carried out on an LC 8A preparative liquid chromatograph equipped with a SPD-M10A VP PDA detector (all Shimadzu). A SunFire C18 column (150 × 30 mm i.d., 5 μm; Waters) connected to a pre-column (10 × 10 mm) was used, at a flow rate of 20 mL/min. HPLC-based activity profiling was performed on an Agilent 1100 system equipped with a PDA detector. A SunFire C18 column (150 × 10 mm i.d., 5 μm; Waters) connected to a pre-column (10 × 10 mm) was used. The flow rate was 4 mL/min. Time-based fractions were collected with a Gilson FC204 fraction collector. ESI-MS spectra were obtained on an Esquire 3000 Plus ion trap mass spectrometer (Bruker Daltonics). NMR spectra were recorded on an Avance III 500 MHz spectrometer (Bruker BioSpin) equipped with a 1-mm TXI microprobe.
Preparative HPLC was carried out on an LC 8A preparative liquid chromatograph equipped with a SPD-M10A VP PDA detector (all Shimadzu). A SunFire C18 column (150 × 30 mm i.d., 5 μm; Waters) connected to a pre-column (10 × 10 mm) was used, at a flow rate of 20 mL/min. HPLC-based activity profiling was performed on an Agilent 1100 system equipped with a PDA detector. A SunFire C18 column (150 × 10 mm i.d., 5 μm; Waters) connected to a pre-column (10 × 10 mm) was used. The flow rate was 4 mL/min. Time-based fractions were collected with a Gilson FC204 fraction collector. ESI-MS spectra were obtained on an Esquire 3000 Plus ion trap mass spectrometer (Bruker Daltonics). NMR spectra were recorded on an Avance III 500 MHz spectrometer (Bruker BioSpin) equipped with a 1-mm TXI microprobe.
5
Size Exclusion Chromatography of Lipid Vesicles
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Here, 100 μL of extract mixture (stained with lipid dye or antibody) was flowed over a SEC column with PBS. Elution fractions were collected into a clear polystyrene 96-well plate (Costar 3370, Corning Inc., USA) at a rate of 0.4 min/well using a Gilson FC 204 Fraction Collector (Gilson, Inc., USA). Poly-Prep chromatography columns (Bio-Rad, USA) were packed with 8 mL of Sepharose 4B resin 45–165 μm bead diameter, (Sigma Aldrich, USA) and washed with sterile PBS 3 times before use. Elution fluorescence was measured using a Synergy H1 microplate reader (BioTek, USA). Excitation and emission wavelengths for SBA-AlexaFluor™ 594 were 590 and 617 nm, respectively. Excitation and emission wavelengths for α-FLAG-DyLight 488 were 493 and 528 nm, respectively. Vesicles stained with FM 4-64 lipid dye were used to determine the characteristic vesicle elution fraction. Reference samples probed with FM 4-64 were used to determine the characteristic vesicle elution fraction in each experiment. For plots, SBA curves were background subtracted.
6
Optimization of Size-Exclusion Chromatography Using Sephadex Beads
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All available size-exclusion dextran-based SephadexTM beads were purchased from Cytiva (formely GE Healthcare, Marlborough, MA, USA) and were as follows: G-10 (17-0010-01), G-15 (170020-01), G-25 fine (17-0032-01), G-50 fine (17-0042-01), G-75 (17-0050-01), and G-100 (17-0060-01). gSEC column was prepared by layering the beads from the smallest to the largest in different Econo-Columns® (Bio-Rad, Hercules, CA, USA) at room temperature by gravity. The 1× or 0.1× phosphate-buffered saline (PBS) was used as the mobile phase. Fractions were collected in Greiner UV-Star® 96-well plates using a FC204 fraction collector (Gilson, Middleton, WI, USA), with 6 drops per well. UV-Vis and fluorescence of the fractions were measured using a Synergy H1 plate reader (Biotek, Winooski, VT, USA).
7
HPLC Analysis of Radioactive Compounds
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The system consisted of two HPLC Kontron 420 pumps (Kontron, Selabo, France), managed with the HPLC software Diamir (Varian Medical Systems, USA). The HPLC system was equipped with a 500 µl loop and an Interchrom Strategy KR100 column C18 (5 µm, 4.6×250 mm) (Interchim, France) kept in an oven thermostated at 35 °C and connected to an on-line radioactivity analyzer (Packard Flo-one, Flow scintillation analyzer) using Flo-scint as the scintillation cocktail or connected to a Gilson FC 204 fraction collector (Gilson, France) for 4 tubes/min collection, using Packard Ultima Gold as scintillation cocktail, when appropriate. Two mobile phases were used. Mobile phase A containing: water/acetonitrile/acetic acid: 97.5/2.5/0.1; and mobile phase B containing: water/acetonitrile/acetic acid: 40/60/0.1. Elution gradient was as follows: 100% A from 0 to 4 min, a linear gradient from 4 to 20 min from 0% to 6% B, a linear gradient from 20 to 25 min from 6% to 25% B, a plateau at 25% B from 25 to 35 min, a linear gradient from 35 to 50 min from 25% to 90% B, a plateau at 90% B from 50 to 60 min.
Urine was injected directly whereas intestinal contents and 0–24 h homogenized feces were extracted by 2 volumes of 0.5 M NaCl.
Urine was injected directly whereas intestinal contents and 0–24 h homogenized feces were extracted by 2 volumes of 0.5 M NaCl.
8
Characterization of Altholactone by NMR and MS
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NMR spectra for altholactone were recorded in DMSO-d6 (δH 2.50 and δC 39.5) at 25 °C on a Bruker AVANCE III HDX 800 MHz NMR spectrometer (Fallanden, Zurich, Switzerland) equipped with a triple resonance cryoprobe. High-resolution electrospray ionization mass spectra (HRESIMS) were recorded on a Bruker maXis II ETD ESI- qTOF (Bruker, Bremen, Germany). The HPLC system for LLE fractions for phenotypic screening included a Waters 600 pump (Milford, MA, USA) fitted with a 996-photodiode array detector and Gilson FC204 fraction collector (Middleton, WI, USA). The HPLC system for purification of re-extracted material was a semi-preparative Thermo Ultimate 3000 system with a PDA detector (Waltham, MA, USA). A Phenomenex C18 Monolithic column (5 μm, 4.6 × 100 mm) was used for LLE fractionation; a Thermo Electron Betasil C18 column (5 μm, 21.2 × 150 mm) was used for semi-preparative HPLC.
9
Nicotine-Evoked Dopamine Release Assay
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Previously described methods [27 (link)] were followed using 7,8-[3H]DA (20–40 Ci/mmol) obtained from Perkin Elmer Life Sciences (Boston, MA). Crude synaptosomal preparations from freshly dissected brain regions were allowed to take up tracer [3H]DA prior to superfusion at 0.7 ml/min for 10 min. Release of DA was stimulated by exposure to nicotine for 20s. Parallel aliquots were exposed to 50 nM α-conotoxin MII (α-CtxMII) (generously provided by Dr. J. Michael McIntosh, University of Utah) for 5 min before the nicotine exposure. Fractions (10 s, ~0.1 ml) were collected into 96-well plates using an FC204 fraction collector (Gilson, Inc., Middleton, WI) for 3.8 min starting one min before nicotine exposure. DA release units are calculated as evoked cpm exceeding baseline cpm, summed and normalized to baseline cpm.
10
NMR and HPLC Characterization of Compounds
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NMR spectra were recorded in DMSO-d6 (δH 2.50 and δC 39.5) at 25 °C on a Bruker Avance HDX 800 MHz spectrometer equipped with a TCI cryoprobe. The HPLC system included a Waters 600 pump fitted with a 996 photodiode array detector and Gilson FC204 fraction collector. All solvents used for extraction and chromatography were Lab-Scan HPLC grade, and the H2O was Millipore Milli-Q PF filtered.
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