Cell counting kit 8 dye

Suramin as a compound that inhibits Sirt5 NAD⁺-dependent deacetylase activity (28 (link)). To determine the optimal concentration of Suramin, BmN cells (1.25×10⁴ cells/well) were pre-incubated with a gradient of increasing concentrations of Suramin (5, 10, 15, 20, 25, 30 and 35 μM, CHEMEGEN, USA) for 24 h in 96 well-plates. Cytotoxicity was determined using Cell Counting Kit-8 dye (Beyotime, China). The optimum concentration was considered the maximal concentration at which Suramin did not cause measurable toxicity to the cells. In all consecutive experiments, BmN cells (1.25×10⁵ cells/well) were pretreated with Suramin at the optimum concentration (30 μM) for 12 h. Cells pretreated with the same volume of solvent (ddH₂O) were used as control. BmN cells pretreated with Suramin or ddH₂O were infected with BmNPV at 1 MOI for 1 h. Then, the supernatant was replaced with Grace medium (10% FBS) supplemented with Suramin (at 30 μM). Total RNA and cell supernatants were collected at 24, 48 and 72 hpi to detect BmNPV replication using qRT-PCR and viral titer assay.

The protocol for cell toxicity has been reported elsewhere77 (link). Cytotoxicity test was determined using Cell Counting Kit-8 dye (Beyotime Inst Biotech) according to the manufacturer’s instructions. Briefly, 5 × 10³ DF-1 cells/well were seeded in a 96-well flat-bottomed plate, incubated at 37 °C for 24 h, and then placed in serum-free conditions for another 6 h. Subsequently, cells were treated with drugs at increasing concentrations in the presence of 2% FBS for 48 h. After 10 μL CCK-8 dye was add to each well, cells were incubated at 37 °C for 2 h and the absorbance was determined at 450 nm using a Multiskan FC microplate reader (Thermo Fisher, Shanghai, China).