Human recombinant full-length MR with His-tag, expressed in mouse myeloma cell line NS0, was purchased from R&D systems, anti-human IgG Alexa-Fluor 488-conjugated from Jackson ImmunoResearch, and anti–penta-His Alexa-Fluor 488 conjugate from Qiagen. Biotinylated Concanavalin A, R. communis agglutinin-I, Maackia amurensis lectin-I, Sambucus nigra lectin, fluorescein Concanavalin A, fluorescein Wheat germ agglutinin, and Man-BSA were purchased from Vector Laboratories; neuraminidase and Protein A-agarose beads from Roche; O-glycosidase, PNGase F, α-galactosidase, β1,3-galactosidase, and β1,4-galactosidase from New England Biolabs; sequencing grade trypsin from Promega; Glucitol-polyacrylamide (PAA), Man3-PAA, and Glucitol-PAA biotin from Lectinity; GlcNAc and GlcNAc-BSA from Carbosynth. All other chemicals were purchased from Sigma-Aldrich if not indicated differently.
Fluorescein concanavalin a
Manufactured by Vector Laboratories
Sourced in United States
Fluorescein Concanavalin A is a fluorescently labeled lectin derived from the jack bean. It binds to glycoproteins containing terminal mannose or glucose residues.
Automatically generated - may contain errors
Lab products found in correlation
Lsm800 confocal microscope, by Zeiss (2 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
Cell culture trypsin egta, by Thermo Fisher Scientific (2 mentions)
Tryple select enzyme, by Thermo Fisher Scientific (2 mentions)
Dnase, by Merck Group (2 mentions)
Facsaria 3, by BD (2 mentions)
Collagenase b, by Roche (2 mentions)
Fluorescein wheat germ agglutinin, by Vector Laboratories (1 mentions)
Biotinylated concanavalin a, by Vector Laboratories (1 mentions)
Sequencing grade trypsin, by Promega (1 mentions)
β1 4 galactosidase, by New England Biolabs (1 mentions)
β1 3 galactosidase, by New England Biolabs (1 mentions)
O glycosidase, by Roche (1 mentions)
Maackia amurensis lectin 1, by Vector Laboratories (1 mentions)
Sambucus nigra lectin, by Vector Laboratories (1 mentions)
Micron 4 system, by Phoenix Pharmaceuticals (1 mentions)
Viscotears liquid gel, by Novartis (1 mentions)
5 protocols using fluorescein concanavalin a
1
Recombinant Human MR Glycosylation Analysis
2
Time-lapse Imaging of Cell Motility
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Dissociated cells were placed in Lab-Tek II coverslip chambers (Thermo Fisher Scientific, Waltham, MA). Time-lapse images with 10 s interval were collected with a LSM 800 confocal microscope with a 63X 1.4 NA objective and fluorescence and DIC optics (Carl Zeiss Microscopy LLC, Thornwood, NY, USA). Fiber cell membranes were counterstained with fluorescein-concanavalin A (# FL-1001, Vector Laboratories, Burlingame, CA, USA). In order to observe fiber cell motility, the glass bottom of the chambers was covered with a thin layer of ECM Gel from Engelbreth-Holm-Swarm murine sarcoma (# E1270, Millipore, Burlington, MA, USA), a substrate that cells adhered to less strongly than to glass.
3
Intravitreal Injection Protocol for Polymer Evaluation
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Adult (6-8 weeks old) C57BL/6J mice (Harlan, Indianapolis, IN) were housed and bred in a Intravitreal injections were performed under a surgical microscope using mice that were anaesthetized with isoflurane (Merial; Animal Health Ltd., Essex, UK). Pupils were dilated using 1% tropicamide and 2.5% phenylephrine (Chauvin, Essex, UK). Viscotears Liquid Gel (Novartis Pharmaceuticals UK Ltd., Surrey, UK) were used to improve the visibility of the fundus. A 33gauge needle (Hamilton Bonaduz AG, Bonaduz, Switzerland) was inserted from the limbus with a 45° injection angle into the vitreous. The direction and location of the needle was monitored through the microscope. One μL of Pull-Et-RhB, Pull-Hy-RhB and Pull-Es-RhB at 500 ng/mL, or 1 μL of PBS, pH 7.4, were injected intravitreally using a repeating dispenser (PB-600-1; Hamilton Bonaduz). Fundus images were taken with the Micron IV system (Phoenix Research Labs, Pleasanton, CA, USA) at 6 and 12 hours after intravitreal injection. The mice received 100 µL of fluorescein concanavalin A (Vector Laboratories, Burlingame, CA, USA) intravenously to label the posterior segment blood vessels and 15 min later, the eyes were collected, retinal flat mounts prepared, and examined by confocal microscopy (Leica SP5, Leica Microsystems, Wetzlar, Germany).
4
Isolation of Choroid Plexus Epithelial Cells
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Mice were euthanized under isoflurane anesthesia, and the CP from all four ventricles of six mice were dissected and collected in 4 °C HBS (in mM: 145.0 Na+, 3.6 K+, 1.8 Ca2+, 0.8 Mg2+, 138.6 Cl−, 0.8 SO42−, 5.5 Glucose, 10.0 HEPES, 2.0 PO42−, with pH 7.4). The pooled CP tissues were incubated in 50 µg/mL Concanavalin A fluorescein (Vector Laboratories, Oxfordshire, GB) in HBS for 10 min at 37 °C, washed and digested in 2 μg/mL dispase (Invitrogen, Carlsbad, CA, USA) and 2 μg/mL collagenase B (Roche, Basel, Switzerland) in calcium-free HBS for 30 min at 37 °C, and incubated in 1:1 mixture of TrypLE Select Enzyme (Thermo-Fisher, Waltham, MA, USA) and cell culture trypsin/EGTA (Thermo-Fisher) supplemented with 1 mg/mL DNase (Sigma, St. Louis, MA, USA) for 10 min at 37 °C. The preparation was inspected on the microscope, passed through a 50 µm filter, and incubated with propidium iodide added before FACS for exclusion of dead cells. Cells were sorted into fluorescein positive and fluorescein negative samples by 4-way purity sorting on a FACSAria III (BD Biosciences, San Jose, CA, USA). The yield and quality control for the FACS isolation of the used CPECs was reported previously [6 (link)].
5
Choroid Plexus Cell Isolation and Sorting
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Mice were anesthetised by isoflurane inhalation and euthanized by cervical dislocation. The choroid plexi (CP) from all brain ventricles were removed and placed in HEPES‐buffered saline (HBS) on ice (Table 1 ). Pooled CP tissues from 6 to 8 mice were incubated in 50 μg/mL concanavalin A fluorescein (Vector Laboratories) in HBS for 10 min at 37°C, digested in 4 μg/mL dispase (Invitrogen) and 4 μg/mL collagenase B (Roche) in calcium‐free HBS for 30 min at 37°C, and incubated in a 1:1 mixture of TrypLE Select Enzyme (Thermo‐Fisher) and cell culture trypsin/EGTA (Thermo‐Fisher) supplemented with 1 mg/mL DNase (Sigma) for 10 min at 37°C. The preparation was passed through a 50‐μm filter, and propidium iodide was added before Fluorescence‐activated cell sorting (FACS) for exclusion of dead cells. Cells were sorted into fluorescein‐positive and fluorescein‐negative samples by 4‐way purity sorting on a FACS Aria III (BD Biosciences). The cells were sorted using a 70‐μm nozzle, at 70 psi and 12–20 kHz. After FACS, the samples as well as control CP samples were spun for 1 min in a table‐top micro‐centrifuge and the pellets were processed for total RNA isolation. Sample purity was validated by RT‐PCR with primers targeting the epithelial marker Claudin‐1 (Cldn1) and the endothelial marker Claudin‐5 (Cldn5).
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