Renilla glo luciferase assay kit

Renilla luciferase expressing sunitinib resistant stable cells (1.5 × 104/well) were seeded in 96-well plate 2 days in advance and then co-cultured with CTLs for 2–3 days. Human peripheral blood CD8+T cells (CTLs) were purchased from StemCell Technology Inc (Vancouver, Canada) and cultured in RPMI media supplemented with 10% FBS, 1% penicillin/streptomycin and 0.1 mg/mL hIL2. They were activated with ImmunoCult™ Human CD3/CD28 T cell Activator (StemCell Technology Inc., Vancouver, Canada) and cultured in a humidified incubator containing 5% CO2 at 37°C for 3–4 days before co-culture with cancer cells. The ratio of cancer cells and CTLs was optimized to 1:2 to 1:4. Sunitinib resistant cells were co-incubated with CTLs alone or CTLs with R428, SB203580 and Metformin at desired concentrations for indicated time. After incubation, luciferase luminescence was evaluated using Renilla-Glo Luciferase assay kit (Promega; Madison, WI) to estimate cancer cell viability. Caspase-Glo 3/7 assay (Promega; Madison, WI) was used to evaluate cancer cell caspase activities or cell death in duplicate plates parallelly.

The Renilla-Glo Luciferase assay kit (Promega) was used to quantify nLuc reporter expression in all knockdown experiments. The buffer volume was reduced to 70 μl of the reconstituted Renilla-Glo luciferase reagent per well of a 96-well plate. To assess nluc reporter expression in the context of Dhx15 transgene expression, the Renilla luciferase assay system (Promega) was used. Briefly, cells were lysed in 120 μl 1x Renilla luciferase assay lysis buffer and 10 μl cell lysate was mixed with 25 μl 1x Renilla luciferase assay substrate. The CellTiter-Glo 2.0 assay (Promega) was used to quantify viable cells, according to the manufacturer`s instructions. Luminescence was measured on a Modulus single tube reader or Perkin Elmer Counter Victor 3 plate reader.

The replication of EBOV in the cells was evaluated by the minigenome system²⁶ . Briefly, producer cells (p0) were cotransfected with p4cis-vRNA-RLuc (250 ng) and pCAGGS-T7 (250 ng) expressing T7 RNA polymerase and four plasmids expressing EBOV proteins (pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), and pCAGGS-L (1000 ng)). One day after transfection, the medium was replaced with a medium containing 5% FBS and then incubated for another 3 days. Target cells (p1 or later) were transfected with pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), and pCAGGS-Tim (250 ng), incubated for 24 h, infected with transcription- and replication-competent virus-like particles (trVLPs) from the p0 (or p1) supernatant for 24 h, and then cultured for another 3 days in medium containing 5% FBS. Viral replication was either determined by intracellular luciferase activities using the Renilla-Glo luciferase assay kit (Promega, E2710) after cell lysis by passive lysis buffer (PLB, Promega) or by viral RNA levels determined by reverse transcription and qRT-PCR.