FITC-Dextran solution (Sigma) was made at a concentration of 25 mg/mL in Milli-Q water. FACS tubes were pre-conditioned at 4°C or 37°C and 2 x 106 DCs were placed in 200 μL of RPMI +10% FBS. 10 μL of the FITC-Dextran solution was added to the tubes and placed either at 4°C or 37°C for 15 min. DCs without FITC-Dextran were incubated at 37°C for 15 min as a control. After 15 min, cells were washed with 1 mL of FACS Buffer (1X DPBS +2% FBS) and spun for 5 min at 1,500 RPM. After spinning, cells were then fixed with 100 μL of 0.5% Formalin (Epredia) for 20 min at room temperature. Cells were washed 2X with FACS buffer and analyzed by flow cytometry using the NovoCyte Quanteon Flow Cytometer (Agilent).
Fitc dextran solution
Manufactured by Merck Group
Sourced in United States
FITC-dextran solution is a fluorescent-labeled dextran product. It is a water-soluble polysaccharide that can be used as a tracer or marker in various biological and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Novocyte quanteon flow cytometer, by Agilent Technologies (1 mentions)
Polycarbonate membrane, by Corning (1 mentions)
Spectramax m5 fluorescence spectrophotometer, by Molecular Devices (1 mentions)
Zθ system, by Applied Biophysics (1 mentions)
Microplate reader, by BMG Labtech (1 mentions)
Synergy h4 hybrid reader, by Agilent Technologies (1 mentions)
Rhodamine 6g, by Merck Group (1 mentions)
Axio imager m1, by Zeiss (1 mentions)
Axiocam hrc, by Zeiss (1 mentions)
Qdot525, by Thermo Fisher Scientific (1 mentions)
Qdot 705, by Thermo Fisher Scientific (1 mentions)
Evans blue, by Merck Group (1 mentions)
Flexstation ii384, by Molecular Devices (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Infinite m200, by Tecan (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
24 protocols using fitc dextran solution
1
FITC-Dextran Uptake Assay in Dendritic Cells
2
FITC-Dextran Permeability Assay
3
Evaluating Endothelial Barrier Function
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Permeability of endothelial monolayers in vitro was determined by FITC-conjugated dextran (FITC-dextran) and transendothelial migration of neutrophils. Briefly, HUVECs or SVECs were seeded on collagen-coated upper chambers (2 × 105 cells/200 μ/well) of transwell inserts (diameter 6.5 mm, pore size 0.4 μm, polycarbonate membrane, Costar); and lower chambers were supplemented with 0.5 ml of culture medium. The cells were cultured for 24 h to allow monolayer form. After pretreatment with different doses of anti-ATP5B antibodies or peptides for 1 h, cells were treated with 20 μg/ml of FlgE or FlgEM for 24 h. Then, 200 μl of FITC-dextran solution (0.5 mg/ml, 40 kDa, Sigma) was added into the upper chambers; and the culture was continued for 2 h at 37°C. Samples were collected from lower chambers, and leaked fluorescence was measured using a fluorescent plate reader. In another experimental setting, a filter with 5.0-μm pore size was utilized for collagen coating, HUVEC growing, and anti-ATP5B antibody treatment. Then neutrophils obtained from peripheral blood of healthy donors were added to the upper chambers (105 neutrophils in 100 μl of medium), and FlgE or FlgEM proteins (20 μg/ml) were loaded to the lower chambers (or not). One hour later, cells that migrated into the lower chambers were countered using a Coulter Counter (BodBoge, Shenzhen, China).
4
Permeability Assay on SIRC Cells
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Permeability assay on filter-cultured SIRC cells was performed by measuring the apical-to-basolateral movements of FITC-dextran solution with average mol wt 70,000 (Sigma–Aldrich, St. Louis, MI, USA) as previously described [50 (link)]. The FITC-dextran solution was added into the apical compartment. Sixty minutes later, the medium from the lower chamber was collected and the absorbance was measured through a microplate reader (Biorad 680, Milan, Italy) using 480 nm for excitation and 535 nm for emission.
5
Intestinal FITC-Dextran Absorption Assay
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Twenty-three hours after IM, 100 μl of fluorescein isothiocyanate (FITC)-dextran solution
(5 mg/ml, Molecular weight: 70,000, Sigma, Tokyo, Japan) was administered to the mice
orally. One hour after oral administration, mice were euthanized, and the intestine from
stomach to the end of the colon was dissected. The intestine was separated into 15
sections, as follows: Sto, stomach; S1–S10, small intestine; C1–C3, colon. The sections
were cut in physiological salt solution (PSS) and then shaken vigorously for 10 sec,
centrifuged at 1,500 g for 5 min at 4°C, and then each supernatant was
transferred to a new tube. The supernatants were then additionally centrifuged at 11,000
g for 5 min at 4°C, and 200 μl of each resulting supernatant was
transferred to wells of a 96-well plate. The fluorescence intensity of FITC was measured
using an EMC-427 plate reader (JASCO Corp., Tokyo, Japan). The ratio of the fluorescence
intensity of each well to the total fluorescence intensity was then calculated. In
addition, the geometric center (GC) of the FITC-dextran distribution was calculated
according to the following equation:
GC=sum (each fluorescence intensity ratio [%] × section number)/100.
(5 mg/ml, Molecular weight: 70,000, Sigma, Tokyo, Japan) was administered to the mice
orally. One hour after oral administration, mice were euthanized, and the intestine from
stomach to the end of the colon was dissected. The intestine was separated into 15
sections, as follows: Sto, stomach; S1–S10, small intestine; C1–C3, colon. The sections
were cut in physiological salt solution (PSS) and then shaken vigorously for 10 sec,
centrifuged at 1,500 g for 5 min at 4°C, and then each supernatant was
transferred to a new tube. The supernatants were then additionally centrifuged at 11,000
g for 5 min at 4°C, and 200 μl of each resulting supernatant was
transferred to wells of a 96-well plate. The fluorescence intensity of FITC was measured
using an EMC-427 plate reader (JASCO Corp., Tokyo, Japan). The ratio of the fluorescence
intensity of each well to the total fluorescence intensity was then calculated. In
addition, the geometric center (GC) of the FITC-dextran distribution was calculated
according to the following equation:
GC=sum (each fluorescence intensity ratio [%] × section number)/100.
6
Paracellular Permeability Assay in Chickens
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In vivo paracellular permeability was assessed by measuring the amount of fluorescently labelled dextrans (FITC-d) that had crossed the digestive epithelium into the blood after oral gavage. Elevated blood levels of this large-size molecule are recognized as an indicator of disrupted tight junctions (TJ)7 (link). According to the animal developmental point, two (d14) or three (d32) chickens per cage subjected to 12 h of feed restriction were randomly selected and orally received FITC-dextran solution (3–5 kDa FITC-d dissolved in saline solution; Sigma Aldrich). An appropriate dose of 8.32 mg/kg of body weight was given as recommended in broiler model41 (link). Blood samples were collected 1 h after FITC-d administration in heparin tubes at the brachial wing vein (n = 8 chickens/treatment at d14 and n = 12 at d32) and temporary stored in a dark-colored container. After centrifugation (1000 g for 10 min at 4 °C), the plasma were collected and diluted in phosphate buffer saline (at 1:2 or 1:6 according to the sample concentration). Plasma FITC-d levels were determined by fluorometric mesureament on the same day (excitation, 485 nm; emission 528 nm; SPARK; TECAN, Männedorf, Switzerland) based on a calculated standard curve (saline solution as a blank).
7
Intestinal EV Trafficking and Permeability
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EVs were incubated with 10 µM DiI (Beyotime Biotechnology, Shanghai, China), a lipophilic fluorescent dye, for 30 min at room temperature. DiI-labeled EVs were isolated using ultracentrifugation and then injected orally into C57BL/6 mice. After 8 h, the mice were killed, and gastrointestinal tissues were acquired. DiI fluorescence and EV distribution were detected by the IVIS spectrum.
Intestinal permeability measurement was performed upon oral gavage of FITC-dextran solution (4 kDa, 600 mg/kg; Sigma-Aldrich). After 4 h absorption and excretion, mice were anaesthetized and exposed to the IVIS Spectrum CT system, and the fluorescent retentions of FITC-dextran were measured.
Intestinal permeability measurement was performed upon oral gavage of FITC-dextran solution (4 kDa, 600 mg/kg; Sigma-Aldrich). After 4 h absorption and excretion, mice were anaesthetized and exposed to the IVIS Spectrum CT system, and the fluorescent retentions of FITC-dextran were measured.
8
Measurement of Intestinal Permeability with FITC-Dextran
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FITC-dextran (FD4) serum concentration was used to assess mice intestinal permeability. All mice were fasted for 4 h, then intragastrically administered 0.2 mL FITC-dextran solution (FITC-dextran, 4 kDa, 40 mg/100 g) (Sigma-Aldrich, USA). All mice were then rested for 5 h without food or water. After anesthesia, blood was collected from orbital sinuses. The blood was then placed into 1.5 mL conical tubes and centrifuged at 3500 rpm for 10 min at 4°C. 75 μL of each serum, in triplicate, were then aliquoted into a 96-well plate. FITC-dextran fluorescence was then measured on a fluorescence plate reader via suitable wavelengths (excitation maximum 488 nm, emission maximum 525 nm) [20 (link)]. The exact FITC-dextran serum concentration was then calculated according to FITC-dextran standard curves.
9
Photochemical Induction of Microvascular Thrombosis
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Microvascular thrombus formation was induced by photochemical injury as described before52 (link). For this purpose, animals received an i.a. injection of a FITC-dextran solution (150 kDa, 2,5%, 6 ml/kg bodyweight, Sigma-Aldrich). 5 min later, the vessel of interest (300 μm in length) was exposed to continuous epi-illumination using the FITC filter cube and appropriate illumination (λ = 488 nm). The field of illumination covers the entire field of view under investigation (as shown in figures and videos; in selected experiments all types of microvessels were epi-illuminated: Video S2 , S3 , S6 ). To assure intergroup comparability, the mean fluorescence intensity was determined in each of the analyzed vessels immediately after onset of light exposure.
10
In Vivo Intestinal Permeability Assay
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The animals underwent in vivo intestinal permeability assay 4 h after the burn injury, according to the method previously described by Costantini et al. [44 ]. Four hours after the burn injury, animals were anesthetized with inhaled isoflurane. A midline laparotomy was performed, followed by location of the cecum and evisceration of a 5-cm segment of the distal ileum with isolation between silk ties. A previously prepared fluorescein isothiocyanate (FITC)-dextran solution (Sigma-Aldrich, St. Louis, MO; 25 mg 4.4 kDa FITC-dextran in 200 μL phosphate-buffered saline solution) was then injected into the lumen of the isolated ileum. The eviscerated intestine was then returned to the abdominal cavity, and the abdominal wall was closed with silk suture. One hour after FITC-dextran injection, blood was collected by cardiac puncture and placed into heparinized Eppendorf tubes for centrifugation at 10,000g for 10 min. The plasma was removed and assayed with a SpectraMax M5 fluorescence spectrophotometer (Molecular Devices, Sunnyvale, CA) to determine the concentration of FITC-dextran. A standard curve for the assay was obtained by serial dilution of FITC-dextran in mouse serum.
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