Briefly, serial dilutions of the viral material was allowed to adsorb on the AV529 cell monolayers at 36°C ± 1°C, 5% ± 2% after which the volume of infection media was adjusted to a suitable volume to allow for incubation at 36°C ± 1°C, 5% ± 2% for 48 hours. After the 48 hour incubation step, the cell monolayers were fixed and stained with a crystal violet (Sigma) and methanol stain and the visible MK-1775 price plaques were enumerated by eye and used
to assign a titre in log10 pfu/ml. The assigned mean infectious titre from 30 independent assays was 1.41 × 107 pfu/ml. Cell culture and infection AV529-19 cells were cultured in DMEM/F12 (Sigma) supplemented with 1% (v/v) Penicillin/Streptomycin (Sigma), 1% heat inactivated ultra-low IgG-FBS (Invitrogen), 1% L-glutamine (Sigma), and maintained in a 37°C incubator in 5% CO2. Prior to each assay, cells were plated one day in advance in 96-well tissue culture plates (Becton Dickinson) ACP-196 molecular weight at a density of 4×104 cells per well in a volume of 200 μl. Next day, plates were visually inspected under a microscope to confirm the cell monolayer was 80-100% confluent.
Serial dilutions of the HSV529 test samples as well as the HSV529 in-house reference control were prepared in culture media. The media from each well was removed, and 50 μl of each viral dilution was added to each well (four replicates were used for each dilution). Afterwards, 50 μl media was dispensed into each infected well for a total volume of 100 μl. 5-FU price Afterwards, 100 μl media was added to the uninfected and negative control wells. The plates were placed at 36 ± 1°C, 5% CO2 incubator for 16 hours. RNA isolation Total RNA was isolated
using total RNA purification 96-well kit (Norgen Biotek). The purified RNA was treated with TURBO DNA-free kit (Applied MS-275 concentration Biosystems) according to manufacture’s instruction. Quantitative real-time RT-PCR (RT-qPCR) The RT-qPCR was performed by targeting the HSV-2 immediate early (ICP27), early (TK) and late (gD2) genes. For ICP27, the forward and reverse primers were 5′- GCC ACT CTC TTC CGA CAC -3′ and 5′- CAA GAA CAT CAC ACG GAA C-3′, respectively. For TK, the forward and reverse primers were 5′-TGG ATT ACG ATC AGT CGC C -3′ and 5′-ACA CCA CAC GAC AAC AAT GC-3′, respectively. For gD2, the forward and reverse primers were 5′-TCA GCG AGG ATA ACC TGG GA-3 and 5′-GGG AGA GCG TAC TTG CAG GA-3, respectively. The ICP27, TK, and gD2 primers have been previously described and tested in other studies. [14–16]. All the primers were purchased from Life Biotechnologies. One step RT-qPCR was performed using SYBR Green PCR master mix (Applied Biosystems), MultiScribe Reverse Transcriptase (50 U/μl, Applied Biosystems), RNase Inhibitor (20 U/μl, Applied Biosystems), 1 pmol of each forward and reverse primer, and 2 μl isolated RNA in a total volume of 25 μl.