Human insulin and FITC-insulin (Human) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Fluorescein isothiocyanate isomer I, 95% (FITC) was purchased from Alfa Aesar (Tewksbury, MA, USA). PD-10 Sephadex™ G-25 M columns were purchased from GE Healthcare (Buckinghamshire, UK). Dulbecco's modified Eagle's medium (DMEM), Hyclon Hank's 1X balanced salt solution (HBSS), penicillin-streptomycin solution and trypsin-EDTA solution were purchased from Fisher Scientific (Middletown, VA, USA). Transwell® inserts were purchased from VWR International (Allison Park, PA, USA). Human insulin ELISA kit was purchased from Crystal Chem (Elk Grove Village, IL, USA). MDCK cell line was purchased from ATCC® (Rockville, MD, USA).
Pd10 sephadex g25 m columns
Manufactured by GE Healthcare
Sourced in United Kingdom
The PD10 Sephadex G25 (M) columns are size-exclusion chromatography columns used for desalting and buffer exchange applications. These columns are designed to separate small molecules from larger biomolecules, such as proteins, peptides, or nucleic acids. The columns are pre-packed with Sephadex G25 Medium, a dextran-based gel filtration medium, and are ready to use.
Automatically generated - may contain errors
Lab products found in correlation
Human insulin, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Transwell inserts, by Avantor (1 mentions)
Human insulin elisa kit, by Crystal Chem (1 mentions)
Mdck cell line, by American Type Culture Collection (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Q sepharose high performance, by GE Healthcare (1 mentions)
Protease inhibitor, by Roche (1 mentions)
4 protocols using pd10 sephadex g25 m columns
1
FITC-Insulin Permeability Assay
2
Multiphoton Microscopy for Renal Hemodynamics
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In order to investigate renal hemodynamics, multiphoton microscopy experiments were performed. Mice were anesthetized by i.p. injections of ketamine/xylazine (100/10 mg/kg of body weight) before surgery was performed on an operating table with a servo-controlled heating plate. The right carotid artery was cannulated using hand-drawn polyethylene tubing so that the arterial blood pressure and the heart rate could be measured continuously. A cannula was inserted into the right jugular vein for the injection of fluorescent dyes and for an intravenous fluid supplementation over the experiment (12 µl/g body weight/h of 0.9% saline). Finally, the left kidney was exposed by making a small flank incision.
Images were then acquired using an inverted Zeiss LSM 710 NLO confocal fluorescence microscope (Carl Zeiss) equipped with a servo-controlled warming plate to maintain the body temperature of the animal at 37 °C. Excitation was achieved using a Chameleon Ultra-II MP laser (Coherent) at 860 nm.
Vasculature was labeled by injecting 80 µl of Texas Red conjugated to 70 kDa dextran (Invitrogen, 20 mg/ml stock solution purified by PD-10 Sephadex G-25M columns (GE Healthcare)) and cell nuclei were visualized using Hoechst 33342 (Invitrogen, 25 µl of a 10 mg/ml solution).
Images were then acquired using an inverted Zeiss LSM 710 NLO confocal fluorescence microscope (Carl Zeiss) equipped with a servo-controlled warming plate to maintain the body temperature of the animal at 37 °C. Excitation was achieved using a Chameleon Ultra-II MP laser (Coherent) at 860 nm.
Vasculature was labeled by injecting 80 µl of Texas Red conjugated to 70 kDa dextran (Invitrogen, 20 mg/ml stock solution purified by PD-10 Sephadex G-25M columns (GE Healthcare)) and cell nuclei were visualized using Hoechst 33342 (Invitrogen, 25 µl of a 10 mg/ml solution).
3
Protein Extraction and Fractionation
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Total soluble proteins from cultured cells were extracted by grinding powdered samples in 10 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, and a cocktail of protease inhibitors (Roche Applied Science, ref. 04693132001). Samples were centrifuged at 16 000 Â g for 20 min at 4 1C and the supernatant was used as a source of soluble proteins. After desalting on PD10 Sephadex G25 (M) columns (GE Healthcare), soluble proteins were fractionated by chromatography on Q-Sepharose High Performance (GE Healthcare) columns (1.6 Â 5 cm) equilibrated with the extraction buffer, and stepwise elution was performed using discontinuously increasing NaCl concentrations in the same buffer (from 0 to 1 M NaCl). Collected fractions were stored at À80 1C for further analyses.
4
Protein Fractionation by AEC
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Preparation of protein extracts and anion exchange chromatography (AEC) fractionation Total soluble proteins from plant shoot and root tissues were extracted by grinding 15 g powdered samples in 10 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, and a cocktail of protease inhibitors (Roche Applied Science). Samples were clarified by centrifugation, desalted on PD10 Sephadex G25 (M) columns (GE Healthcare), and 30-40 mg soluble proteins were then fractionated by chromatography onto Q-Sepharose High Performance columns (1.6 x 5 cm), as described previously (Sarthou et al., 2020) (link). Stepwise elution was performed using discontinuous increasing NaCl concentrations in the extraction buffer (from 0 to 1 M NaCl; Sub-fractions F0 to F1000). The salt concentrations were chosen to obtain a homogenous repartition of the quantity of proteins in each sub-fraction (Supplementary Figure S1). Collected sub-fractions were stored at -80°C until used for further analyses. Four independent fractionation experiments from independent 8-plant cultures each were performed (Series 1 to 4).
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