Milli q water mq

The possible antiviral activity of the compounds was investigated in Vero cell lines using each compound in a non-toxic concentration. Cells were seeded in 96-well plates 4 × 10⁴ cells/well and infected at multiplicity of infection (MOI) of 0.01. After 1 h incubation, the cells were washed with PBS to remove the unattached HSV-2 and the PBS was replenished with a medium containing 2-fold dilutions of compounds, each starting with the first non-toxic concentration. Acyclovir was used as a positive control in the same concentration as the compounds. After 24 h incubation at 37 °C by 5% CO₂ concentration, the cells were washed and suspended in Milli-Q water (MQ) (Millipore, Billerica, MA, USA). After 2 thaw–freeze cycles, the cells were resuspended. Using HSV-2 specific gD2 primers F: 5-TCA GCG AGG ATA ACC TGG GA-3′, R: 5-GGG AGA GCG TAC TTG-CAG GA-3, qRT-PCR was performed to check the HSV-2 genome quantity, as described earlier [27 (link)]. Measurement was performed with Bio-Rad CFX96 (Bio-Rad, Hercules, CA, USA). Selectivity index was calculated as described earlier (SI = (CC₅₀/IC₅₀) [28 (link)].

Proteoliposomes
were immobilized on a passivated glass surface in home-built chambers
and imaged by TIRF microscopy. Chamber parts were cleaned extensively
by using ethanol and Milli-Q water (MQ; Millipore). Glass slides (thickness
170 ± 10 μm) were cleaned by consecutive rounds of sonication
by 2% (v/v) Helmanex following three washes (×3) with MQ and
×2 with methanol. Glass slides were dried in nitrogen flow, plasma
etched for 2 min, mounted in a microscope chamber, and incubated with
a mixture of 1000:6 PLL-g-PEG and PLL-g-PEG-biotin (SuSoS, Switzerland) (1 g/L) in surface buffer (15 mM
HEPES, pH 5.6) for 30 min. After carefully washing with a sample buffer
(20 mM HEPES, 100 mM NaCl, pH 7.5), we incubated the surfaces with
0.1 g/L neutravidin (Life Technologies) in the surface buffer for
10 min after additional washing with sample buffer. Proteoliposome
surface density was controlled by addition of 4 μL (0.05 g/L)
proteoliposomes to an 80 μL chamber volume. The chamber was
washed × 10 times in a sample buffer when the desired surface
density reached. Before imaging, sample buffer containing 2.5 mM protocathechuic
acid (PCA), 50 nM protocatechute-3,4-dioxygenase (PCD), and 1 mM 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Trolox) was injected into the chamber.

The antiviral activity of phenanthrenes was investigated in Vero cells. Cells were seeded in 96-well plates and were infected with HSV-2 at a multiplicity of infection (MOI) of 0.01. After a 1 h adsorption period, the inoculum was removed, the cultures were washed twice, and culture medium containing the plant compounds in different concentrations was added. After a 24 h incubation period, the cultures were washed with phosphate buffered saline, and finally 100 μL Milli-Q water (MQ) (Millipore, Billerica, MA, USA) was added to the cells, and the cultures in the plate were frozen.