Endothelial permeability measurements were performed using a Transwell system with HMEC-1 cells seeded in the upper chamber with a 5.0 µm pore size (CLS3421, Corning, USA) until they reached 90% confluence. Freshly isolated CXCR4lo and CXCR4hi neutrophils from healthy controls and psoriasis patients (1×105/well) were added to the upper chamber and co-incubated at 37 °C for 6 h. LDHA-IN-3 (50 μM, MCE) was added to block lactate activity. Transwell inserts without stimulation or with DMSO were used as controls. After 6 h, the culture medium was removed and washed with free-cell medium three times. Next, 50 µl of FITC-labeled dextran (D1844, 2.5 mg/mL, 40 kDa, Invitrogen) was added to the upper chamber as a tracer. After 2 h, 100 µl samples were collected from the lower chamber and fluorescence spectrophotometry was performed with an excitation wavelength of 494 nm and an emission wavelength of 521 nm (spectrofluorometer, Varioskan LUX 3020-265, Thermo Scientific) to measure the permeability of the endothelial monolayer.
D1844
Manufactured by Thermo Fisher Scientific
Sourced in United States
The D1844 is a high-performance centrifuge from Thermo Fisher Scientific. It is designed for a wide range of applications in research and clinical laboratories. The centrifuge provides reliable and consistent results with its advanced rotor design and precise speed and temperature control capabilities.
Automatically generated - may contain errors
4 protocols using d1844
1
Measuring Endothelial Permeability in Psoriasis
2
Measuring Endothelial Cell Permeability
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TEER was measured using a EVOM voltohmeter connected to an Endohm 9 electrode chamber (World Precision Instruments, Sarasota, FL) as previously described [29 (link)]. Tissue culture inserts containing monolayers of HGEc cultured on Transwell-Collagen-coated membrane, were placed in the chamber and the TEER was recorded after 10 sec [29 (link)]. The permeability experiments were done as described before [31 (link)]. Briefly, 3 x 105 HGEC-1 were plated onto Transwell Collagen-coated membrane inserts (Corning Costar, Cat No 3415) in DMEM media with 10% FBS, and left for 3 days to form mature monolayer with a trans-endothelial electrical resistance (TEER) of ~ 60 ± 10 Ω /cm2. Subsequently, the cells were starved for 5 hours in serum free DMEM media without phenol red, treated with the corresponding reagents, and incubated with 1 mg/ml FITC-dextran (Molecular Probes, D-1844, Invitrogen) for 30 min at 37°C. Samples collected from the bottom chambers were read in triplicates on the Victor 3V1420 multi-counter (Perkin-Elmer, Wellesley, PA).
3
pSIVA-IANBD Probe for RGC Axon Phosphatidylserine Detection
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The fluorescent probe pSIVA linked to N,N′-di-methyl-N(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyleneamine (pSIVA-IANBD; NBP2-29382; Novus Biologicals, Centennial, CO, USA) was used to detect phosphatidylserine (PS) externalization along the RGC axonal membrane.8 (link) Intravitreal injections of the probe were performed under the surgical microscope immediately posterior to the superotemporal limbus. After the sclera was penetrated with a 30-gauge needle at a 45° angle, a 32-gauge needle was attached to a 5 µL Hamilton syringe (Hamilton, Reno, NV, USA) for injection. This location of puncture and entry route of administration helps avoiding both retinal detachment and injury to the lens. A 4 µL volume was slowly injected over a 60-second period. The needle was held in place for another 60 seconds, then slowly withdrawn. The injection perforation was sealed by application of small amount of surgical tissue adhesive glue (3M Vetbond, St. Paul, MN, USA). Dextran-fluorescein of 40,000 MW (catalog D1844; ThermoFisher, Hillsboro, OR, USA) was used as a non-PS binding control for injections.
4
Paracellular Permeability Assay
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After the TEER assay, dextran solution (10 mM HEPES [pH 7.4], 1 mM sodium pyruvate, 1 mM CaCl2, 10 mM glucose, 145 mM NaCl, 1 mM MgCl2, 0.33 mg/mL fluorescein-labeled dextran, 40 kDa [D1844; Thermo Fisher Scientific], 0.33 mg/mL Texas Red-labeled dextran, 3 kDa [D3329; Thermo Fisher Scientific]) was added to upper chamber. After 2 h of incubation at 37 °C, the solution of lower chamber was collected, and fluorescence intensity was measured using microplate reader (Ex 488 nm/Em 528 nm and Ex 590 nm/Em 615 nm).
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