The wound healing and tube formation assays were performed as described previously [29 (link)]. Briefly, a monolayer of cells was scratched and imaged at zero time and after 18 h. Images were acquired using an Axiovert 200 microscope (Carl Zeiss). Images were analysed for the percentage of migration. For the tube formation assay, equal numbers of cells were grown in 3D Matrigel overnight and imaged after 18 h. Images were analysed for mean tube count per field. For permeability assay, BMVECs were cultured in Transwell plates (Corning Life Sciences, Acton, MA, USA) for 24 h in complete medium and then in serum-free medium for an additional 24 h. the confluent monolayer of BMVECs in the upper chamber of the Transwells was treated with FITC–dextran (1 mg/ml, mol. wt 150,000; Sigma). The fluorescence intensity, equivalent to the relative amount of FITC–dextran in the lower chambers of the Transwells, was measured over a 30 min period and determined using a Biotek Spectrometer (Biotek Instruments, Winooski, VT, USA) (excitation wavelength, 485 nm; emission wavelength, 530 nm). Experiments were performed three times in duplicates.
Fitc dextran
Manufactured by Corning
Sourced in United States
FITC-dextran is a fluorescent labeling agent used in various laboratory applications. It consists of the fluorescent compound fluorescein isothiocyanate (FITC) covalently attached to dextran, a polysaccharide derived from the bacterium Leuconostoc mesenteroides. FITC-dextran is utilized as a tracer molecule for studying permeability, diffusion, and other biological processes in research settings.
Automatically generated - may contain errors
Lab products found in correlation
Fitc dextran, by Merck Group (2 mentions)
Axiovert 200 microscope, by Zeiss (1 mentions)
Transwell plate, by Corning (1 mentions)
Fitc dextran, by Agilent Technologies (1 mentions)
0.4 μm polycarbonate transwell membranes, by Corning (1 mentions)
Black walled 96 well microtiter plate, by Corning (1 mentions)
E plate 16, by Roche (1 mentions)
Envision 2104 multilabel plate reader, by PerkinElmer (1 mentions)
Black bottom 96 well plate, by Corning (1 mentions)
Sephadex g 100, by Merck Group (1 mentions)
4 protocols using fitc dextran
1
Assessing Angiogenic and Permeability Properties
2
Transwell Permeability and Real-Time Cell Analysis
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For FITC-dextran flux, cells were seeded (5 × 104 cells/well) onto FN-coated (10 μg/ml) 0.4-μm polycarbonate Transwell membranes (Corning) and cultured for 48 h. FITC-dextran (10 kDa) (Sigma-Aldrich) at a final concentration of 1 mg/ml was added to the upper chamber, and after 2 h, medium from the bottom chamber was collected and transferred to a black-walled, 96-well microtiter plate (Corning) for analysis. Fluorescence intensity was analyzed using a plate reader (Tecan; excitation 485 nm, emission 520 nm). For RTCA experiments, 5 × 104 cells were plated in each well of an E-Plate 16 (Roche Applied Science), and cell index was assessed for 30 h.
3
Assessing Caco-2 Monolayer Permeability
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Permeation to uncharged molecules was assessed via unidirectional 4 kDa FITC-dextran (Sigma, Burlington, MA, USA) flux. After incubation of filter-grown Caco-2 monolayers with 13-HPODE, monolayers were washed with PBS and supplied with fresh phenol red free-serum free medium. Thereafter, 1 μg/mL FITC-dextran was added to the top chamber in which 100 μL aliquots were removed from the bottom well and added to a black-bottom 96-well plate (Corning, New York, NY, USA) in triplicates at indicated time points. An equal amount of medium was returned to the bottom wells to preserve volume. Fluorescence was measured in a PerkinElmer Envision 2104 Multilabel Plate Reader at an excitation wavelength of 490 nm and emission wavelength of 520 nm. For consistency, fluorescent flux values obtained 30 min after addition of dextran to top well were used.
4
Synthesis and Characterization of Magnetic Nanoparticles
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Fluorescein isothiocyanate diethylamino ethyl (FITC-DEAE)-Dextran (M w 40 000 Da), FITC-Dextran (M w 40 000 Da), dextran (from leuconostoc M w 40 000 Da), ferric chloride hexahydrate FeCl 3 •6(H 2 O) (ACS reagent, 97%), ferrous chloride tetrahydrate FeCl 2 •4(H 2 O) (reagentplus®, 98%), ammonia hydroxide solution (28-30%), Corning® Spin-X® (100k), sodium azide, and sephadex® G-100 were all purchased from Sigma Aldrich. Spectra/Por® Biotech cellulose ester (CE) dialysis membranes (MWCO: 100 000) were purchased from Fisher Scientific. Deionised water was used from a Milli-Q system (resistivity 15 MΩ cm at 25 °C). Millex GP syringe filters with a polyethersulfone (PES) 0.22 µm membrane were purchased from Fisher Scientific.
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