Celltiter glo luminescent kit

Drug sensitivity assay was performed as described previously [10 (link)]. Briefly, cells were seeded in 96-well plates (12,000 cells per well) in 0.1 ml medium and treated for 72 h with serially diluted anticancer drugs. CellTiter-Glo (CellTiter-Glo Luminescent kit, Promega) reagents (50 μl) was added to each well and mixed for 10 min before the luminescence was measured on a microplate reader (Biotek, USA).

Intracellular ATP was determined by CellTiter-Glo® Luminescent Kit (G7571, Promega Heidelberg, Germany) according to the manufacturer’s protocol. After indicated drug treatment, medium was replaced with the new one, and the reagent in the kit was added to lyse the cells. The plate was gently shaken for 2 min, placed stationary in the dark for 10 min, and then 150 μl solution was transferred to a 96-well plate for luminescence detection using LB96V MicroLumat plus (American Laboratory Trading, Boston, USA). With background subtraction, the values were normalized to individual control group as 100 %.

Duplicates of five-fold serial dilutions of the four polymers were tested in human lung tissue (HLT) cells using at least three different donors. HLT cells were added at a density of 300,000 cells/well and incubated with the compounds for 1 h before infection. Then, a MOI of 0.1 of the VSV^*ΔG(Luc)-S virus was added to the wells, and plates were spinoculated at 1,200 g and 37°C for 2 h. After the infection, fresh RPMI medium was added to the wells and cell suspensions were transferred into a 96-well flat-bottom plate. Cells were then cultured overnight at 37°C in a 5% CO₂ incubator. Each plate contained the following controls: no cells (background control), cells treated with medium (mock infection), cells infected but untreated (infection control) and cells infected and treated with the drug camostat mesylate (S2874, Sigma) as a positive control (Grau-Expósito et al., 2022 (link)). After 20 h, cells were incubated with Britelite plus reagent (Britelite plus kit; PerkinElmer) and then transferred to an opaque black plate. Luminescence was immediately recorded by a luminescence plate reader (LUMIstar Omega). In parallel, drug cytotoxicity was monitored by luminescence. To evaluate cytotoxicity, the CellTiter-Glo Luminescent kit (Promega), was used. Data were normalized to the mock-infected control, after which EC₅₀ and CC₅₀ values were calculated.

Cells were seeded in white 384-well plates at 1000 cells/well for both viability and caspase 3/7 assays. Drugs were added at the indicated concentrations 24 h post seeding. After 72 h of drug treatment, viability and caspase 3/7 were determined using the CellTiter-Glo Luminescent Kit (Promega: G7570) and Caspase-Glo 3/7 Assay Kit (Promega; G8091), respectively, according to the manufacturer’s instructions. Caspase signals were normalized to the amount of viable cells in corresponding conditions in the same experiment.

Cells were plated in 96-well plates and 2 d later were serum-starved for 24 h. Cells were then incubated with conditioned media and/or drugs for an additional 48 h before cell viability/proliferation assays were performed using the CellTiter-Glo® Luminescent kit (Promega) according to the manufacturer’s instructions.