Celltiter glo luminescent substrate

Tacrolimus (FK506) (Selleckchem), pimecrolimus (Selleckchem), sirolimus (Selleckchem), and ionomycin (Alomone labs) were all dissolved in DMSO (Sigma Aldrich). Activity of the compounds on VZV gB/gH-gL mediated cell fusion were evaluated by stable reporter fusion assay as described previously [28 (link)]. Briefly, CHO-DSP1 or MeWo-DSP1 cells transfected with equal quantities of pCAGGS-gB, pME18S-gH[TL], and pcDNA3.1-gL plasmids using Lipofectamine 2000 were harvested at 6 hrs post-transfection, and mixed with MeWo-DSP2 cells in the presence of various concentrations of the compounds prepared in two-fold serial dilutions, ranging from 10 μM to 1.25 μM. Co-culture of cells were seeded into Nunc MicroWell 96-well Optical-Bottom Plates (ThermoFisher) and incubated for 48 hrs. The activity of Renilla luciferase was read immediately after adding substrate h-Coelenterazine (Nanolight Technology). Transfection with only vehicle plasmids pcDNA3.1 (+) and pME18S, or pcDNA3.1 (+), pME18S and pCAGGS-gB served as negative control. Cell viability was measured using CellTiter-Glo Luminescent substrate (Promega). Luminescence signal was recorded using Synergy H1 Hybrid Multi-Mode Reader (BioTek). Experiments were performed at least in triplicate.

C6/36 cells (1 × 10⁵) were seeded in a 96-well plate and infected at an MOI of 5 PFU/cell. At designated time points, CellTiter-Glo luminescent substrate (Promega) was added to quantify the number of viable cells according to the manual. Luminescence was recorded using a Wallac Victor³ 1420 Multilabel counter (PerkinElmer, Waltham, MA, USA).