Amicon ultra 15 pltk ultracel pl membrane 30 kda

Fluorescent DENV was prepared by labeling with Alexa Fluor 594 succinimidyl ester (AF594SE, Molecular Probes, Invitrogen, Carlsbad, CA, USA) according to the method described in a previous study^{36 (link)}. The labeled viruses were purified using Amicon Ultra-15 PLTK Ultracel-PL Membrane (30 kDa) centrifugal filter units (Millipore) to remove excess dye. After labeling, the DENVs were tested for fluorescence and stored in 50 μl aliquots at −80 °C prior to use.

BV2 immortalized murine microglial cells, obtained from Dr. C. C. Huang (Department of Pediatrics, National Cheng Kung University, Tainan, Taiwan), were grown in Dulbecco’s Modified Eagle’s Minimal Essential Medium (DMEM; Invitrogen Life Technologies, Rockville, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen Life Technologies), 50 U/ml penicillin, and 50 μg/ml streptomycin in a humidified atmosphere with 5% CO₂ and 95% air. Baby hamster kidney (BHK) cells and Aedes albopictus C6/36 cells were cultured in DMEM containing FBS. C6/36 cell-adapted DENV serotypes (DENV1 8700828, DENV2 PL046 and 454009A, DENV3 8700829A, and DENV4 59201818) were obtained from the Centers for Disease Control in Taiwan and maintained accordingly43 (link). Fluorescent DENV particles were prepared by labeling with AlexaFluor 594 succinimidyl ester (AF594SE, Molecular Probes, Invitrogen, Carlsbad, CA) according to a previous study44 (link). The labeled viruses were purified using Amicon Ultra-15 PLTK Ultracel-PL Membrane (30 kDa) centrifugal filter units (Millipore, Billerica, MA) to remove excess dye. The entry of labeled DENV into cells was analyzed using a fluorescent microscope (BX51; Olympus, Tokyo, Japan) and quantified using a FACSCanto II Flow Cytometer (BD Biosciences, San Jose, CA).

Fluorescent DENV was prepared by labeling with Alexa Fluor 594 succinimidyl ester (AF594SE, Molecular Probes, Invitrogen) as referred to the previous studies [21 (link)]. The labeled viruses were purified using Amicon Ultra-15 PLTK Ultracel-PL Membrane (30 kDa) centrifugal filter units (Millipore) to remove excess dye. Cells were washed twice after an inoculation (MOI = 1) with cells for 2 h at 37°C. Cells were visualized under a laser-scanning confocal microscope (Leica TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany) and were analyzed using FACSCanto II Flow cytometer (BD Biosciences, Franklin Lakes, NJ). The three-dimensional images reconstructed from a series of confocal images, along with the z-axis of the cells and the analysis of z-stacks, were reconstructed using the Leica Confocal Software.