Verification of the reactivity of the two functionalities of the ester-alkyne and N-hydroxysuccinimide esters (NHS-ester) was conducted by reacting fluorescently labeled molecules with the two groups. Specifically, Alexa Fluor® 555 azide (500 µg mL−1, Yao-Hong Biotechnology, Taiwan) was used to detect the ester-alkyne group on the multifunctional poly-para-xylylene nanoparticles. A conjugation reaction, an azide-alkyne cycloaddition/click reaction, was expected without a copper catalyst, and the reaction was conducted at 25 °C in an aqueous solution (pH of 7.4) for 2 h. A centrifugation rinsing process (14,000 rpm) using deionized water as a resuspension agent was performed afterwards to remove any unreacted Alexa Fluor® 555 azide. For the NHS-ester group on the multifunctional poly-para-xylylene nanoparticles, the detection was conducted using FITC-labeled BSA (2 mg mL−1, Sigma Aldrich, USA), and the conjugation reaction through the amine/NHS−ester coupling reaction was performed at 25 °C in an aqueous solution for 4 h. Finally, the same centrifugation rinsing process was used to remove any unreacted FITC-labeled BSA.
Fitc labeled bsa
Manufactured by Merck Group
Sourced in United States, Germany
FITC-labeled BSA is a fluorescent conjugate of bovine serum albumin (BSA) and the fluorescent dye fluorescein isothiocyanate (FITC). It is commonly used as a tracer or marker in various biological applications, such as cell labeling, immunoassays, and protein detection.
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Lab products found in correlation
11 protocols using fitc labeled bsa
1
Verification of Ester-Alkyne and NHS-Ester Reactivity
2
Quantifying Albumin Uptake in MSCs
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Cellular uptake of albumin was analyzed in confluent MSC monolayers using fluorescein isothiocyanate (FITC)-labeled albumin (Sigma-Aldrich), as previously described [32 (link)]. Briefly, after 14 days of co-culture with RPTECs or stimulation with EVs or TOT-CM, cell medium was refreshed and MSCs were incubated with 50 μg/ml of FITC-labeled BSA (Sigma-Aldrich) for two hours at 37°C. Fluorescence emission was measured by cytofluorimetric analysis.
3
Measuring Endothelial Cell Permeability under Hyperglycemia
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To evaluate the effects of hyperglycemia on endothelial cell proliferative ability, cell permeability was tested using the BSA-FITC kit [11 (link), 12 (link)]. HRMECs were seeded in the upper Transwell-COL membrane insert (24 wells, Corning Incorporated, NY, USA). After reaching 90% confluence, the HRMECs were starved for 12 h in serum-free medium and subsequently replaced with control or experimental medium for 24 h. After 24 h, a tracer solution of FITC-labeled BSA (250 μg/ml; Sigma-Aldrich, Germany) was added to the upper insert, and 500 μl medium without FITC-BSA was added to the lower insert. After incubation for 2 hours, the collected media were measured using a fluorophotometer (Tecan, Switzerland) at an emission/excitation wavelength of 495/520 nm.
4
Protein Adsorption Evaluation on Implants
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FITC-labeled BSA (Sigma) in PBS was used for the protein adsorption test. Adsorption was measured using fluorescence intensity. FITC-conjugated serum albumin was added in order to examine plasma protein adsorption by the SA, CA, and SOI implants. After incubation, the excess FITC-conjugated serum albumin was washed from the implant surfaces. Three implants of each surface were treated with FITC-conjugated serum albumin, examined using a Typhoon FLA 7000 device (GE Healthcare Life Sciences, Chicago IL, USA), and analyzed using Image J software (National Institute of Health, Bethesda, MD, USA) (Figure 2 ).
5
Podocyte Albumin Permeability Assay
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Differentiated podocytes were seeded on transwell bicameral chambers (0.4 μm pore; Corning, Cambridge, MA, USA) and transepithelial passage of FITC-labeled BSA (Sigma) was measured. Confluent differentiated podocytes were exposed to palmitate for 90 min in Ca2+-free KRB solution. In the upper compartment of transwell chambers, culture medium was replaced with 2 ml of FITC-BSA (250 μg/ml). From the lower compartment 100 μl samples were drawn after 0.5, 1, 2, 3 and 4 h of diffusion. The chamber was replenished with an equal volume fresh medium each time a sample was withdrawn. The fluorescence intensity of collected samples was measured using a fluorescence microplate reader (Flexstation II) at 450 nm, and the albumin concentrations in the samples were calculated using linear regression.
6
Fluorescent Microstructure Fabrication
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FITC-labeled BSA (Sigma Aldrich), AlexaFluor 647-labeled BSA (AlexaFluor), and DOX (LifeTein LLC) were simultaneously added to the photoresist formulation to a concentration of 1 mg mL−1 each. The photoresists were directly transferred to printing with the previous printing parameters without any additional step. The printed microstructures were thoroughly rinsed to remove non-polymerized photoresist and were imaged in a Nikon Ti-E fluorescence microscope.
7
Characterizing Nanomedicinal Macromolecules
8
Quantifying Vascular Barrier Permeability in GEnCs
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The permeability of GEnC monolayers was determined using Costar Transwell plate with 0.5-μm porous filters and FITC-labeled BSA (Sigma-Aldrich, Darmstadt, Hessen, Germany), as described previously (37 (link)). GEnCs were grown on the upper chamber of Costar Transwell until confluent. The tracer protein FITC-albumin was added to the upper chamber after relevant stimulation. After incubation at 37°C for 30 min, samples from both the upper and lower chambers were collected for fluorometric analysis. Fluorescent intensity (FI) was measured using a microplate fluorescence reader (TristarTM LB941, Berthold, Germany) with filter settings of 485 nm (excitation) and 538 nm (emission). Eventually, these fluorescence readings were used for calculation of the permeability coefficient, which is indicative of vascular barrier disruption. The permeability coefficient was calculated according to the following formula:
Permeability coefficient = FI (lower chamber) × 100% / (FI (upper chamber) + FI (lower chamber)).
Permeability coefficient = FI (lower chamber) × 100% / (FI (upper chamber) + FI (lower chamber)).
9
Measuring Vascular Permeability in GEnC Monolayers
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GEnC monolayer permeability was determined with fluorescein isothiocyanate (FITC)-labeled BSA (Sigma), as described previously [24 (link)]. Cells were grown on Costar Transwell 0.4-μm porous filters (Coming, Acton, USA) until confluent. Also, in order to demonstrate the effect of moesin in this process, the cells were preincubated with blocking antibody to moesin before treatment with anti-MPO antibody, MPO-ANCA-positive IgGs and/or HMGB1. After treatment, the tracer protein FITC-albumin was added to the luminal chamber for 45 min, and samples were taken from both the luminal and abluminal chambers for fluorometry analysis. Fluorescence signals were measured in a microplate fluorescence reader (Tristar™ LB941) with filter settings of 485 nm (excitation) and 538 nm (emission). These readings were then used to determine the permeability coefficient of albumin which could stand for the vascular barrier disruption. All data are shown as the ratio of control at the corresponding test.
10
Fluorescent Microstructure Fabrication
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