After respective treatments, mice were injected with 16.7 mg/kg FITC-Dextran 10 kDa (FD10S, Sigma-Aldrich) 1 h before euthanasia (Egawa et al., 2013 (link)). Brains were collected, homogenized in 50 mM Tris-Cl (pH = 7.6; 1 μl/mg brain) and centrifuged at 16,000 g for 30 min at 4°C. Fifty microliter of supernatant was transferred to a 96 well black polystyrene assay plate (Corning 3915) for analysis. A series of standards containing 0.005, 0.02, 0.1, 0.5, 2.5, 5 and 10 μg/ml FITC-Dextran 10 kDa in 50% Tris-HCl/50% absolute methanol were used. The concentration of FITC was determined by spectrofluorometry with an excitation of 485 nm (20 nm bandwidth) and an emission wavelength of 528 nm (20 nm bandwidth).
Fd10s
Manufactured by Merck Group
Sourced in United States
The FD10S is a freeze dryer manufactured by Merck Group. It is designed for the lyophilization of samples, providing a controlled environment for the removal of water or other solvents from a variety of materials. The FD10S features a stainless-steel chamber, a temperature-controlled shelf, and a high-performance vacuum system to facilitate the freeze-drying process.
Automatically generated - may contain errors
Lab products found in correlation
Black bottomed 96 well plate, by Corning (2 mentions)
Varioskan lux, by Thermo Fisher Scientific (2 mentions)
Hpds o ir pulsed laser, by Dotmatics (2 mentions)
X3 ol ir pulsed laser, by Dotmatics (2 mentions)
Fd40s, by Merck Group (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Phenol red free dmem, by Thermo Fisher Scientific (2 mentions)
Sodium acetate, by Thermo Fisher Scientific (1 mentions)
Hplc grade, by Thermo Fisher Scientific (1 mentions)
Urethane, by Merck Group (1 mentions)
Dpx mounting medium, by Thermo Fisher Scientific (1 mentions)
Tetracaine base bp grade, by Merck Group (1 mentions)
Dapi medium, by Merck Group (1 mentions)
Hydrogen peroxide 30, by Avantor (1 mentions)
Ethanol, by Merck Group (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Soluene 350, by PerkinElmer (1 mentions)
A9771, by Merck Group (1 mentions)
Fitc albumin, by Merck Group (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
21 protocols using fd10s
1
FITC-Dextran Brain Permeability Assay
2
Detailed Experimental Materials Catalogue
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Acetonitrile and methanol both HPLC grade, DPX mounting medium, xylene and Optiphase scintisafe gel were purchased from Fischer Scientific (Leicester, UK). Tetracaine base BP grade (99.9%), formalin solution neutral buffer 10%, DAPI medium, ethanol, heparin sodium salt (I-A), urethane, 0.7% glacial acetic acid, isopropanol and FTIC-dextran with average molecular weight (Mw) of 4 kDa (FD-4) and 10 kDa (FD-10S) used without any further purification steps were supplied by Sigma Aldrich (Dorset, UK). Concentrated hydrochloric acid and sodium hydroxide was from Fluka (Dorset, UK). Sodium acetate was provided by Alfa Aesar (Heysham, UK). Silicone membranes with a thickness of 0.25 mm were purchased from GBUK Healthcare (Selby, UK). Phosphate buffered saline (Dulbecco A) tablets were obtained from Oxiod Limited (Hampshire, England). The Tissue-Tek® O.C.T™ compound, scintillation vials and hydrogen peroxide 30% were obtained from VWR International (Leicestershire, UK). Dextran (carboxyl-14C) with an average M.W. of 10 kDa and specific activity of 0.00006 Ci/mmol was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, USA). Soluene® 350 was provided by Perkin Elmer (Bucks, UK). Isoflurane 100% (w/v) inhalation vapour liquid was obtained from Animal Care Ltd. (York, UK).
3
Transepithelial Permeability Assay
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After the TER measurement, the basal medium was replaced with phenol red-free DMEM (#21063-029; Thermo Fisher Scientific) supplemented with 5% FBS. The apical medium was replaced with medium containing 1 mg/ml of FITC-dextran with a molecular mass of 10 kD (FD10S; Sigma-Aldrich). After incubation at 37°C for 2 h, the fluorescence intensity of the basal medium was measured using a microplate reader (VARIOSKAN LUX; Thermo Fisher Scientific) in a black-bottomed 96-well plate (Corning). The apparent permeability coefficient (Papp) was calculated using the following equation (Watson et al., 2001 (link); Van Itallie et al., 2008 (link)): where dQ [mg] is the amount of tracer transported to the basal acceptor compartment during incubation time dt [s], A [cm2] is the area of the filter and C [mg/cm3] is the initial concentration of the tracer in the apical donor compartment.
4
Assessing Retinal Vascular Leakage in Mice
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To assess retinal vascular leakage, pups were anesthetized using isoflurane and a mixture of 10 kDa-FITC-dextran, 100 mg/mL (Sigma, FD10S) and fluorescent Alexa568-conjugated IB4 (5 mg/kg) was injected retro-orbitally into EC-Foxc1-KO and littermate control P6 pups (2.8−4.1 g), EC-Foxc1-KO and littermate control P21 pups (5.9−12.05 g) and Pericyte-Foxc1-KO and littermate control P6 pups (2.4−3.9 g). The volume of the combined injections of dextran and IB4 was determined based on the body weight of each pup. Euthanization was performed after 10 min of circulation and eyes were enucleated. A small pinch was made in the cornea to ensure sufficient fixation by PFA. Fixation was performed in 4% PFA for 20 min. at RT. Dissection was performed in 4% PFA and retinas were incubated in 4% PFA/PBS for 2 h at room temperature followed by washing with PBS and mounting on glass slides.
5
Dextran Relocation as LMP Indicator
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LNCaP cells were grown on tissue culture dishes with cover glass bottom (FluoroDish, FD35, World Presicion Instruments, Inc.). Two days after plating cells were incubated with FITC-dextran (10 kDa, 1 mg/ml) (FD10S, Sigma) for 7 h, washed and chased for 3 h in complete medium. Then cells were treated with vehicle, FQ (20 μM) or CQ (20 μM) for 12 h and dextran relocation was analyzed by confocal microscopy. In control non-treated cells FITC-dextran appears in puncta, consistent with its reported localization in lysosomes. LMP provokes release of FITC-dextran from lysosomes to cytosol thereby increasing cytoplasmic fluorescence intensity.
6
Evaluating Endothelial Cell Permeability
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ECs (5×105) were seeded on the insert of a Transwell filter (Coring 3460, 0.4 μm pore size) in 24-well dishes and grown 2–3 days until they reached confluence. After treatment with LPS (1 μg/ml), FITC–dextran (FD10S, Sigma, USA) was added to the upper chamber at a final concentration of 1mg/mL. After 30 min of incubation at 37°C, 50 μL samples were taken from the lower chamber of each Transwell and transferred to a 96-well plate for fluorescence measurements. The fluorescent content of the samples was measured using a fluorescence plate reader optically filtered for 485nm excitation and 535 nm emission [30 (link)].
7
Vascular Permeability Quantification
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Vascular permeability in the scrambled siRNA treated and Gja1 siRNA treated groups was assessed repeatedly using a albumin-fluorescein isothiocyanate (FITC) conjugate tracer (A9771, FITC-albumin, 66 kDa, Sigma, St. Louis, MO) and a fluorescein isothiocyanate-dextran tracer (FD10S, FITC-dextran, 10 kDa, Sigma, St. Louis, MO). Around 100 μl tracers (40 mg/ml) were intravenously administered to the tail vein of anesthetized control siRNA treated and Gja1 siRNA treated groups for 30 min prior to transcardial perfusion of the mice with PBS. After circulating blood cells were completedly flushed away, the stria vascularis was gently emoved. The tissues were then homogenized in 1% Triton X-100 in PBS, with the lysate centrifuged at 13,000 rpm for 20 min. The fluorescence signal, reflecting concentration of fluorescence tagged tracer trapped in the stria vascularis, was detected with a Tecan GENios Plus microplate reader at an excitation wavelength of 450 nm, with the emission acquired through a 560-nm filter.
8
Visualizing Airway Responses in LPS-stimulated Mice
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Cd11c-mCherry mice were stimulated with LPS (30 µg in 40 µL PBS, delivered intranasally as a single dose, 5 days prior to experimentation). Explants from these mice were then cultured in DMEM/F12 containing 1 mg/ml 10Kd FITC-dextran (Sigma FD10S) and 10 µM of methacholine (Sigma A2251). Imaging was conducted on the Olympus FVMPE-RS multiphoton laser scanning microscope equipped with a MaiTai HPDS-O IR pulsed laser (800 nm for FITC) and INSIGHT X3-OL IR pulsed laser (1050 nm for mCherry) with 1024×1024 scan size and a step size 0.5 µm. A 15× optical zoom and line averaging scan were used for high-resolution imaging.
9
Measuring Epithelial Barrier Permeability
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After TER measurement, the basal medium was replaced with phenol red-free DMEM (Life Technologies) supplemented with 10% FBS. The apical medium was replaced by the medium with 1 mg/ml FITC-dextran of molecular mass of 3–5 kDa (FD4; Sigma-Aldrich), 10 kDa (FD10S; Sigma-Aldrich), or 250 kDa (FD250S; Sigma-Aldrich). The cells were incubated in a 5% CO2 incubator at 37°C for 2 h, and the basal medium was collected. Fluorescence intensity of the medium was measured by microplate reader equipment (VARIOSKAN LUX; Thermo Fisher Scientific) in a black-bottomed 96-well plate (Corning). Standard curves were determined by measuring the fluorescence intensities of a serial dilution series of the FITC-dextran. Blank measurement of the medium without FITC-dextran was subtracted from the sample values. The apparent permeability coefficient (Papp) was calculated using the following equation:
where dQ is the amount of tracer transported to the acceptor (basal) compartment during the time period dt, A is the area of the filter, and C is the initial concentration of the donor (apical) compartment.
where dQ is the amount of tracer transported to the acceptor (basal) compartment during the time period dt, A is the area of the filter, and C is the initial concentration of the donor (apical) compartment.
10
Endocytosis dynamics in breast cell lines
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Endocytosis differences between MCF7, T47D, and MCF10-A cell lines were measured using FITC-dextran (FD10S, average molar weight: 10.000; Sigma-Aldrich Co.). Cells were grown in 24-well plates over poly-L-lysine-coated coverslips for 12 hours, then treated with FITC-dextran at 0.5 mg⋅mL−1 and incubated for 0, 6, and 12 hours in culture conditions. After washing with cold PBS and fixing with 4% paraformaldehyde, the coverslips were mounted onto slides, using mounting medium with 4′,6-diamidino-2-phenylindole (DAPI). The samples were analyzed by fluorescence confocal microscope and quantification was performed using ImageJ 1.47t software (National institutes of Health, Bethesda, MD, USA).
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