The dye-coupling assay to test the functionality of GJs was performed using carboxyfluorescein (2187, Sigma-Aldrich, USA) and Dextran AlexaFluor 568 (10,000 MW, D22912, Invitrogen, USA) was used as a control. Chondrocytes were seeded on plates (28 cm2 culture plate) and cultured until the confluence reached 100%. Cells were rinsed two times with PBS and then two scrapes were made using a scalpel in the presence of 0.5% (w/v) carboxyfluorescein and 0.5% (w/v) Dextran AlexaFluor 568 (D22912, Invitrogen, USA) in DMEM at room temperature. Cells were incubated for 2 min at room temperature. After washing with a DMEM, dye transfer was captured using AXIOCAM MRm ZEISS - HBO100 (Zeiss, Germany) with Axioplan 2 (Zeiis, Germany) with Axiovision 4.6 (Zeiss, Germany) software. The number of dye-positive cells (carboxyfluorescein transfer) from the cutting site (red/green cells) to the farthest visual uptake of carboxyfluorescein (only green cells) indicates the GJ connectivity between cells. The score was calculated as previously reported [3 ].
Carboxyfluorescein
Manufactured by Merck Group
Sourced in United States
Carboxyfluorescein is a fluorescent dye used in various laboratory applications. It is a derivative of fluorescein, a well-known fluorescent compound. Carboxyfluorescein is commonly used as a tracer or label in biological and biochemical experiments, providing a way to visualize and track molecules or cells of interest.
Automatically generated - may contain errors
Lab products found in correlation
Carboxyfluorescein, by BMG Labtech (4 mentions)
Fluostar optima, by BMG Labtech (4 mentions)
Transwell, by Corning (2 mentions)
Chloroform, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Axiovision 4, by Zeiss (1 mentions)
Dextran alexafluor 568, by Thermo Fisher Scientific (1 mentions)
Sm2 bio beads, by Bio-Rad (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Sephadex g 75, by Merck Group (1 mentions)
Chloroform, by Thermo Fisher Scientific (1 mentions)
L α phosphatidylethanolamine pe, by Merck Group (1 mentions)
Indocyanine green icg, by Thermo Fisher Scientific (1 mentions)
Vivotrack 680, by PerkinElmer (1 mentions)
1 2 distearoyl sn glycero 3 phosphocholine dspc, by Avanti Polar Lipids (1 mentions)
1 2 distearoyl sn glycero 3 phosphoethanolamine n methoxy polyethylene glycol 2000 ammonium salt dspe m peg2000, by Avanti Polar Lipids (1 mentions)
18 0peg2000pe, by Avanti Polar Lipids (1 mentions)
Sulforhodamine, by Merck Group (1 mentions)
Luciferase, by Creative Biomart (1 mentions)
Multiporator, by Eppendorf (1 mentions)
24 protocols using carboxyfluorescein
1
Dye-Coupling Assay for Gap Junction Functionality
2
Quantitative Analysis of Peptide Uptake by Liposomes
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TmrAB-containing liposomes (0.4 µM) were incubated with C4ATTO655 peptides (1 µM), ATP or ADP (3 mM each), MgCl2 (5 mM), and CPFs (1 µM) for 5 min at 45°C. Transport reactions were stopped by adding EDTA (10 mM). Samples were diluted to a final TmrAB concentration of 20 nM with transport buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 5% [v/v] glycerol) and analyzed by flow cytometry (FACSCelesta). For regression analysis, liposomes were loaded with increasing amounts of peptides to convert the fluorescence intensities into the number of peptides per liposome (Stefan et al., 2020 (link)). For this, liposomes were destabilized by adding Triton X-100 while increasing the C4ATTO655 concentration. Equal amounts of carboxyfluorescein (Sigma-Aldrich) served as loading control. Detergent was removed by adding SM-2 Bio-beads (Bio-Rad) as detailed above. Liposomes were harvested for 30 min at 270,000 g and washed three times by centrifugation. Generally, 20,000–100,000 proteoliposomes were selected according to sideward and forward scatter areas. Single events were selected based on the height of forward scatter plotted against the area of forward scatter. Mean fluorescence intensities of fluorescein and ATTO655 were calculated using FlowJo 10.6.1 software. Data represent mean ± SD from three experiments.
3
Measuring Transwell Monolayer Permeability
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Permeability of the TM cell monolayer was measured using carboxyfluorescein as previously described with minor modification [24 (link)25 (link)26 (link)27 (link)]. Briefly, primary cultured human TM cells were incubated in the inner chamber (insert diameter, 12 mm; pore size, 0.4 µm) of a 12-well plate (no. 3460, Transwell; Corning, Lowell, MA, USA) supplemented with 10% fetal bovine serum. After the cells reached confluence, the media was changed to 1% serum containing DMEM to avoid the effects of growth factors and proteins. Then, the TM cells were exposed to each drug for 24 hours. After washing, 50 µM of the tracer carboxyfluorescein (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. The media was collected from the outer well to analyze fluorescence after 2 hours, and the concentration of carboxyfluorescein in the collected media was measured using a spectrofluorometer (FLUOstar Optima; BMG Labtech, Offenburg, Germany) with an excitation wave-length of 490 nm and an emission wavelength of 530 nm.
4
Lipid-Based Nanoparticle Formulation
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1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] ammonium salt (DSPE-m-PEG2000 or 18:0PEG2000PE), and cholesterol were bought from Avanti Polar Lipids LLC (Alabaster, AL, USA), L-α-Phosphatidylethanolamine (PE) from egg yolk was purchased as a lyophilized powder from Sigma Aldrich (St. Louis, MO, USA). All lipids purchased were used without further purification. SN-38 in powder was purchased from Fisher Scientific (Waltham, MA, USA) and its stock solution was prepared in dimethyl sulfoxide (DMSO; 10 mg/mL). Reagents such as methanol and chloroform were purchased from Fisher Scientific and used as provided. Sephadex G-75 and carboxyfluorescein were purchased from Sigma Aldrich (St. Louis, MO, USA). Indocyanine green (ICG) was purchased from Fisher Scientific (Waltham, MA, USA). Vivotrack 680 was purchased from PerkinElmer (Hopkinton, MA, USA).
5
Fluorescent Labeling of Biomolecules
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Unless stated otherwise, chemicals were from Sigma-Aldrich and were the highest grade available. Unbranched FITC-labeled polyethylene glycols (PEGs) were purchased from Creative PEGWorks (27516). Carboxyfluorescein and sulforhodamine were from Sigma-Aldrich. Other fluorophores were from Invitrogen. Luciferase was from Creative Biomart. Purified GFP was a generous gift from Michael Rosen (UT Southwestern, Dallas, TX, USA).
6
Optimizing Cell Uptake of Contrast Agents
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To determine optimal parameters, a range of electroporation conditions was evaluated for NSCs and ECs using carboxyfluorescein (Sigma) as a proxy agent in hypo-osmolar buffer in 2 mm cuvettes using a Multiporator (Eppendorf). It was evident that uptake into NSCs was highly influenced by voltage, whereas uptake into ECs was subject to pulse length (Supplementary Fig. 2 ). To achieve an efficient uptake of paraCEST agents into these cell types, these two parameters (voltage and pulse length) were adjusted. Notably, for Eu-HPDO3A uptake into NSCs, voltages of 530 V, 650 V and 750 V were applied with a pulse length of 100 μs. In contrast for Yb-HPDO3A uptake into ECs, a 530 V voltage was combined with pulse lengths of 100, 300 and 500 μs. As uptake into either cell type is also dependent on cell densities, these were arrayed at 3, 6 and 9 × 106 cells/mL. Cells were plated overnight before being washed 3x HBSS and harvested using Accutase.
7
Plant-based Protein Emulsion Preparation
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Plant emulsions were made by suspending 5% (w/w) plant protein in reverse osmosis (RO) water and mixing at room temperature for 15 min. Coconut oil was heated to 30 °C and 5% (w/w) was added to the plant protein suspension and pre-homogenised using a rotor-stator homogeniser (Polytron PT3100, Kinematica AG, Malters, Switzerland), prior to homogenisation at 30/300 bar (Panda 2k, GEA NIRO Soavi, Parma, Italy). The pH of the emulsions was set to 6.8 using 10% lactic acid prior to autoclaving at 105 °C for 30 min. Plant emulsions for determining acidification were supplemented with 0.5% (m/v) via a 20% glucose stock and sterile 1% 1.064 mM carboxyfluorescein (Sigma-Aldrich, St. Louis, MO, USA) as described earlier [11 (link)]. To minimise the amount of rich medium transferred into the various plant emulsions during inoculation with bacterial strains, the inoculum prepared in rich medium were diluted with plant suspensions. For this, plant protein suspensions per protein type were created by suspending 5% (w/w) plant protein into RO water and mixing at room temperature for 15 min. The pH of the suspensions was adjusted to 6.8 using 10% lactic acid prior to autoclaving at 105 °C for 30 min. After cooling on ice, the suspensions were supplemented with 0.5% (m/v) via a 20% glucose stock.
8
Transwell Assay for TM Cell Permeability
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A permeability study of the TM cell monolayer was performed as previously described with minor modification [13 (link)14 (link)15 (link)16 (link)17 (link)]. Briefly, primary cultured human TM cells were incubated in the inner chamber (insert diameter, 12 mm; pore size, 0.4 µm) of a 12-well plate (no. 3460, Transwell; Corning, Lowell, MA, USA) at 2 × 104 cells/mL supplemented with 10% FBS. After the cells reached confluence, the media was changed to 1% serum-containing Dulbecco's modified Eagle media to avoid the effects of growth factors and proteins in serum. Then, the TM cells were exposed to each drug for 24 hours. After washing three times with PBS, 50 µM of the tracer carboxyfluorescein (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. The media was collected from the outer well to analyze fluorescence after 2 hours, and the concentration of carboxyfluorescein in the collected media was measured using a spectrof luorometer (FLUOstar OPTIMA; BMG Labtech, Offenburg, Germany) with an excitation wavelength of 490 nm and an emission wavelength of 530 nm.
9
Biomimetic Alginate-Chitosan Nanoparticles
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Medium viscosity sodium alginate (80,000–120,000 Da), low molecular weight chitosan (50,000–190,000 Da), TPP, calcium chloride, acetic acid, Nile-red, carboxyfluorescein, silver nitrate, and PBS were purchased from Sigma-Aldrich (St Louis, MO, USA). BSA and BCA protein assay kit was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA).
Human adipose stem cells were obtained from the Research Institute of Dental Sciences, Shahid Beheshti University of Medical Sciences, Iran, Tehran. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and Dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemicals (Germany). Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal Bovine Serum (FBS), and Penicillin–Streptomycin 1% were obtained from Gibco (Grand Island, NY, USA). All aqueous solutions were prepared using double distilled water. All materials for Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) were prepared as previously reported. All reagents used were of analytical grade.
Human adipose stem cells were obtained from the Research Institute of Dental Sciences, Shahid Beheshti University of Medical Sciences, Iran, Tehran. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and Dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemicals (Germany). Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal Bovine Serum (FBS), and Penicillin–Streptomycin 1% were obtained from Gibco (Grand Island, NY, USA). All aqueous solutions were prepared using double distilled water. All materials for Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) were prepared as previously reported. All reagents used were of analytical grade.
10
Oligomeric Calcitonin Interactions
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Human calcitonin (>95%) was obtained from GL Chemicals Ltd. (Shanghai, China). Mag, Hon and EGCG were from Aladdin-reagent (Shanghai, China). Anti-oligomer antibody (OC), anti-fibril antibody (A11) and anti-rabbit IgG were obtained from Merck Millipore (Billerica, USA). Thioflavin-T (ThT), 2-Oleoyl-1-palmitoyl-snglycero-3-phospho-rac-(1-glycerol) sodium salt (POPG) and carboxyfluorescein were from Sigma-Aldrich (St. Louis, USA). SH-SY5Y cells were obtained from the China Center for Type Culture Collection (CCTCC). All other chemical reagents were of the highest grade available.
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