Cell proliferation assay kit

The adenovirus neutralization assay was performed as previously described (24 (link)), except a newly synthesized HAdV7-Luc virus was used. In brief, HAdV7-Luc was incubated with 3-fold serial dilutions of heat-inactivated sera (starting at 1:12 and ending at 1:8,748) for 1 h at 37°C before 2 × 10 (4 (link)) A549 cells were added to 96-well plates. Simultaneously, diluent without serum was used as a negative control. After incubation for 24 h at 37°C, the inoculum was removed, and luciferase expression was detected using a Luciferase Assay System (Promega, Madison, WI). The reciprocal of the dilution multiple corresponding to a 90% reduction in the fluorescence value (IC₉₀) compared to that of the negative control was designated as the nAb titer, using non-linear regression with four parameters in GraphPad.
In addition, a traditional cytopathic effect (CPE) assay was conducted to validate the stability, reliability, and accuracy of the HAdV7-Luc-based neutralization assay. A mixture of HAdV7-Luc and serum was incubated with HEK293 cells for 6 days before cell viability measurement using a Cell Proliferation Assay kit (Promega, Madison, WI). The correlation of these two methods was then analyzed using Spearman correlation analysis.

The MTS assay was performed to detect viable cell numbers using a cell proliferation assay kit from Promega (Madison, WI, USA). For the MTS assay, MDA-MB-231 and BT549 cells were seeded in 96-well-plates at 2 × 10³ cells/well, cultured for 24 h in growth media, and then treated with CORO2A siRNA for 24, 48, 72, and 96 h. The absorbance (A) at 490 nm was detected using a microplate reader.

The proliferation rates were measured using a Cell Proliferation Assay kit (Promega) according to the manufacturer's instructions. Briefly, 3×10³ cells/well were seeded on 96-well plates and allowed to grow. After the indicated time, 20 µL of substrate solution was added and the cells were incubated for 1 h to allow color development. The absorbance at 492 nm was measured to indicate the number of proliferating cells.

The mouse monocyte/macrophage cell line, RAW 264.7 (ATCC TIB-71), and human colon cancer cell line, SW480 (ATCC CCL-228), were purchased from ATCC (ATCC, Manassas, VA, USA) and cultured and treated as previously described by Dey et al. [16 (link)] and Liu et al. [5 (link)], respectively. Cells were grown in DMEM supplemented with 10% FBS, 1% penicillin (25 U/mL)/streptomycin (25 μg/mL) in a 95% air/5% CO₂-humidified atmosphere at 37 °C. Briefly, RAW264.7 cells were treated with PEITC or DMSO (as a negative control) at a pre-determined dose for 6 h before elicitation with 1 μg/mL of LPS without IFNγ priming. SW480 cells were pretreated with IFNγ 10 ng/mL or control medium (as a negative control) for 12 h, and treated with PEITC or DMSO for 5 h and then stimulated with LPS 10 ng/mL for 4 h. PEITC treatments were performed at 5, 10 and 15 μM concentration. Relative number of viable cells were measured using Cell Proliferation Assay kit (MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; Promega, Madison, WI, USA) following the manufacturer’s instructions.