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Lucifer yellow dye

Manufactured by Merck Group
Sourced in United States

Lucifer yellow dye is a fluorescent marker used in biological research. It functions as a labeling agent to visualize and track cellular structures and processes.

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11 protocols using lucifer yellow dye

1

In Vitro Model of BBB for OE-MSC Transmigration

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To analyze OE-MSC transmigration, we used a model of BBB with two compartments separated by a porous membrane (pore size: 1 μm), already described by Molino et al. [32 (link)]. Briefly, rat endothelial cells from blood cerebral microvessels were cultured in the top compartment on polyethylene insert filters coated with collagen IV and fibronectin, at a cell density of 500,000 per insert in 6-well culture plates. Rat glial cells were cultured in the bottom compartment at a density of 16,000 cells/cm2. For both compartments, the culture medium was composed of DMEM/Ham F12 supplemented with 20% bovine serum depleted in platelets, 2 ng/mL fibroblast growth factor 2 (FGF2), and 500 nM hydrocortisone [32 (link)]. For each experiment, 3 to 4 conditions were evaluated in duplicate: 0, 120,000, and 500,000 OE-MSCs were seeded on top of the endothelial cells, then incubated for 24 h. Permeability to the Lucifer yellow dye (LY, Sigma-Aldrich L0259) was next measured over 60 min using fluorimetry, as described [32 (link)].
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2

Assessing Intercellular Communication via Gap Junctions

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Intercellular communication via gap junctions was characterized using dye transfer after scrape loading, as described before [28 (link)]. In brief, confluent HUVEC monolayers in six-well plates were used. Three cuts were applied with a surgical blade in each well after washing with PBS containing Ca2+ and Mg2+ three times. Thereafter, cells were loaded with lucifer yellow dye (Sigma-Aldrich, Darmstadt, Germany) by incubation of the cells in a warmed dye solution (1 mg/mL) at room temperature for 5 min in the dark. After this period, the dye solution was rigorously washed away and the HUVECs were fixed using 10% formalin. Fluorescence and bright field images of the individual scratches were immediately acquired thereafter using the motorized inverted fluorescence microscope described above. Images were taken using a 10× objective and exposure times of 1/7 or 1/1900 s and stored on a hard disk at a resolution of 1360 × 1024 pixels for analysis later. The area covered by stained cells was determined as the readout of dye diffusion through gap junctions using ImageJ. Hitherto, the images were converted to black-and-white using a threshold function and the area was measured after calibration.
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3

Visualizing Epidermal Lucifer Yellow Uptake

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Lucifer yellow dye (1 mg/mL; Sigma-Aldrich, USA) was topically applied on the epidermis for 1 h. Excess Lucifer yellow was washed away with 1 × PBS, and the skin tissues were cryofrozen with OCT tissue freezing medium (Leica, USA). Tissues were cryosectioned at 8 μm and mounted with Hoechst 33,342 dye (Life Technologies, USA). Images were taken using the LSM 710 confocal microscope with the Zeiss EC Plan-NEOFLUAR 20×/0.5 NA objective. Analyses were performed with ZEN 2012 Light Edition software (Carl Zeiss, Germany).
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4

Measuring Gap Junction Intercellular Communication

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The SL/DT assay was conducted to measure GJIC using Lucifer yellow dye migration through all connexin channels with the scrape and load technique [34 (link)]. After cells were washed 3 times with Dulbecco’s phosphate-buffered saline (D-PBS) without Ca2+ and Mg2+, 0.05% Lucifer yellow dye (Sigma-Aldrich) dissolved in D-PBS was added to cells and six scrapes were made with a surgical steel blade. After 9 min of incubation at room temperature in the dark, the Lucifer yellow was discarded. Cells were washed 3 times with D-PBS and fixed with 4% formaldehyde solution. The GJIC activity was analyzed by monitoring the extent of diffusion of the fluorescent dye Lucifer yellow from the scrape line into adjacent cells through functional gap junctions using an inverted fluorescent microscope (IX61, Olympus, Tokyo, Japan).
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5

Quantifying Epidermal Barrier Permeability

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Fifty microliters of 1 mmol/L Lucifer yellow dye (Sigma-Aldrich) was added to the epidermal surface of the organotypic culture and incubated at 37°C for 4 hours. Metal cloning rings were used to control uniform dosing on the epidermal surface. Lucifer yellow was removed, and the organotypic cultures were washed in PBS before formalin-fixed paraffin embedding under standard conditions. Four-micrometer sections were deparaffinized, counterstained with 4′-6-diamidino-2-phenylindole dihydrochloride (1 μg/mL for 10 minutes; Life Technologies, Carlsbad, Calif), and imaged with a confocal Zeiss LSM710 microscope (Zeiss, Oberkochen, Germany). Quantification of dye penetration in the upper dermis (average intensity in the upper 40 μm) was performed with Zeiss Zen software and compared by using paired t tests.
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6

Evaluating Functional Gap Junctions In Vitro

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For functional gap junction evaluation in vitro, the scratch assay was conducted as previously described.[53 (link)
] Briefly, astrocyte cultures were grown to confluency before the assay. Cells were rinsed with CaMg‐PBS (phosphate‐buffered saline). 1 mg mL−1 solution of Lucifer yellow dye (L0259; Sigma) was added to cover the cell monolayer, and the cells were scraped using a surgical blade. After 1 min incubation at room temperature, the cells were washed thoroughly with PBS and re‐incubated with culture medium, then observed under a fluorescence microscope.
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7

Evaluating Chondrocyte Gap Junctions

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The functional gap junctions between chondrocytes were evaluated with a scrape loading and Lucifer yellow transfer assay. Briefly, cells were seeded in dishes and grown to confluency. The medium was discarded, and cells were rinsed with CaMg‐PBS three times. A 1 mg/mL solution of Lucifer yellow dye (L0259; Sigma) was added to cover the cell monolayer, and the cells were scraped using a surgical blade. After a 5‐10‐minute incubation at room temperature, the cells were washed thoroughly with PBS and fixed by adding 4% paraformaldehyde. The cells were imaged with CLSM (Olympus & Nikon) to determine the number of fluorescent cells with Lucifer yellow uptake which is a measure of gap junction intercellular communication (GJIC).
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8

Immunostaining and Western Blot Analysis

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BAK, dimethyl sulfoxide (DMSO), and Triton X-100, collagenase I and Lucifer Yellow dye were purchased from Sigma Aldrich (St. Louis, MO); Protein A/G PLUS-Agarose Immunoprecipitation Reagent was from Santa Cruz Biotechnology (Santa Cruz, CA); PVDF Western Blotting Membrane was from Roche (Basel, Switzerland); pentobarbital sodium was from Abbott Laboratories (North Chicago, IL); Enhanced chemiluminescence (ECL) kit was obtained from GE Healthcare UK (Chalfont, UK); Mounting medium with 4, 6-diamidino-2-phenylindole (DAPI) and bovine serum albumin (BSA) were from Vector Laboratories (Burlingame CA); Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies (Carlsbad, CA); mouse-anti-rabbit ZO-1 antibody, Alexa488-conjugated donkey-anti-mouse IgG, and Alexa555-conjugated donkey-anti-goat IgG were from Life Technologies (Carlsbad, CA); goat polyclonal antibody for Cx43 and P-Cx43, Horseradish peroxidase (HRP)-conjugated donkey anti- goat IgG were from Santa Cruz Biotechnology (Santa Cruz, CA); mouse anti-rabbit β-actin antibody from Sigma Aldrich (St. Louis, MO); Horseradish peroxidase (HRP)-conjugated goat anti- mouse IgG from Merck (Darmstadt, Germany).
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9

Evaluating Cellular Junctions and Signaling

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DMSO and Triton X-100 were purchased from Abcone (Shanghai, China). Pentobarbital sodium, TNF-α, and Lucifer yellow dye were obtained from Sigma-Aldrich (St. Louis, MO). Primary ZO-1 antibody (mouse anti-rabbit), donkey anti-mouse IgG Alexa Fluor 488, and donkey anti-goat IgG Alexa Fluor 555 were purchased from Thermo Fisher Scientific (Carlsbad, CA). Santa Cruz (Santa Cruz, CA) provided goat-anti-rabbit Cx43 antibodies; HRP-conjugated donkey anti-goat IgG was from Bio-Rad (Hercules, CA). HUABIO (Hangzhou, China) supplied mouse anti-rabbit β-actin antibody, whereas Bio-Rad supplied HRP-conjugated goat anti-mouse IgG (Hercules, CA). Magnetic protein G beads and protein A/G beads were from Millipore (Billerica, MA) and Bimake (Houston, TA) ,repectively. PVDF membrane and 0.25% Trypsin-EDTAwere obtained from Thermo (Carlsbad, CA); ECL kit was provided by Shanghai Epizyme Biomedical Technology Co., Ltd (Shanghai, China); DAPI-mounting media were provided by Vector Laboratories (Burlingame, CA); BSA powders and collagenase I were acquired from Sangon Biotech (Shanghai, China) while DMEM, DMEM/F12, FBS, and Penicillin-Streptomycin (PS) were obtained from Thermo (Carlsbad, CA).
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10

Lucifer Yellow and FM4-64 Endocytosis Assays

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For Lucifer yellow assays, cells were grown in liquid YE5S culture with or without latrunculin A, spun down, and resuspended in YE5S (25 μl) containing Lucifer yellow dye (10 μl of 40 mg/ml; Sigma-Aldrich; made up in water and stored in the dark at 4°C) with or without latrunculin A. Cells was incubated on a tabletop rotor for 30 min at room temperature. Cells were washed two times with 100 μl of potassium phosphate buffer (50 mM potassium phosphate, pH 7.5, 10 mM NaF, 10 mM NaN3) and imaged immediately on a Zeiss scanning confocal microscope (Zeiss, Jena, Germany). For FM4-64 assays, cells were grown in liquid YE5S culture with or without latrunculin A for 10 min, stained with 20 mM FM4-64 (Molecular Probes, Eugene, OR) for 1 min at room temperature, washed with YE5S, and imaged after 10 min.
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