DENV serotype 2 local Taiwan strain 454009 A and ZIKV Asian strain PRVABC were propagated in C6/36 cells as previously described [51 (link)]. To prepare high titers of DENV, we concentrated DENV-containing culture supernatants with Macrosep Advance Centrifugal Devices (molecular weight cutoff of 30 kDa; Pall Corp., Port Washington, NY, USA) at 6000×g at 4°C and stored below −70°C until use. UV-inactivated DENV (UV-DENV) was obtained by treatment with UV 100 mJ/cm2 for 100 s in a UV-crosslinker (VILBER, France). To deplete NS1, we incubated DENV-containing culture supernatant with mouse anti-dengue NS1 1D33-agarose (Leadgene) at 4°C for 1 h, followed by centrifugation at 3000×g at 4°C to obtain NS1-depleted DENV supernatant. The virus titer and NS1 concentration in each condition were analyzed by fluorescent focus assay and NS1 ELISA after the process.
Uv crosslinker
Manufactured by Vilber
Sourced in France
The UV crosslinker is a laboratory equipment used to expose samples to ultraviolet (UV) radiation. It is designed to induce cross-linking in nucleic acids or proteins, a process that is essential for various applications in molecular biology and biotechnology.
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Lab products found in correlation
6 protocols using uv crosslinker
1
DENV and ZIKV Propagation and Purification
2
Oxidative Stress Measurement in UV-Irradiated Fibroblasts
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Normal human dermal fibroblast (NHDF) cells were plated and incubated at a density of 1.0 × 106 cells/well in six-well culture plates for 24 h. The following day, cells were UV irradiated with 40 J/m2 followed by treatment with UV crosslinker (Vilber Lourmat) for 3 min and either untreated or treated with RG3 extract for 24 h. Cells were washed, stained with 5 μM 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Life Technologies, Carlsbad, CA, USA) and 20 mM dihydroethidium (DHE) in Hank's balanced salt solution and incubated at 37°C for 30 min. FACSCalibur flow cytometer (BD Biosciences) was used to measure H2O2 activity using an emission filter at 532 nm. At least 10,000 cells were analyzed in three independent experiments.
3
Mitochondrial Membrane Potential Assay
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NHDF cells were irradiated with 40 J/m2 UV followed by treatment with UV crosslinker (Vilber Lourmat) for 3 min and either untreated or treated with 10 μM of RG3 extract for 24 h or 30 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 8 h as a control. Cells were stained with 1 μM rhodamine-123 for 60 min to measure the mitochondrial transmembrane potential using flow cytometry. A minimum of 10,000 cells per sample were analyzed.
4
UV-Induced Stress in Keratinocytes
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Human keratinocyte HaCaT cells (2 × 104 cells/mL) were grown in 24-well plates for 24 hr. For UV exposure, the medium was removed from the dish, and then culture medium without FBS was added to the dish. UV irradiation (UVA 4 J/cm2) was supplied by UV cross-linker (Vilber Lourmat, France) equipped with 6 × 8 W tubes which emits most of their energy with an emission peak at 365 nm (UVA). After irradiation, cells were incubated in RPMI 1640 medium with 10% FBS.
5
Cell Migration Assay with UV Stress
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For the cell migration assay, monolayers were carefully scratched using a 10-μL pipette tip. After 24 hr of UV irradiation (40 J/m2) and treatment with UV crosslinker (Vilber Lourmat, Collégien, France) for 3 min, cells were untreated or treated with 1, 5, or 10 μM RG3 extract for 24 h. Wounded areas were then photographed.
6
Cellular Wound Healing Assay with Rh2 Extract
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To perform cellular wound healing assays, monolayers of cells were scraped using a 10 μl pipette tip. After 24 h of irradiating (80 mJ/cm2) the cells with UV with a UV crosslinker (Vilber Lourmat, Collegien, France), the cells were un treated or treated with Rh2 extract at a concentration of 1, 10, or 50 μM for 24 h. Thereafter, the wound site was photographed using a microscope.
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