Edu apollo 488 in vitro imaging kit

An EdU Apollo^® 488 In Vitro Imaging kit (Guangzhou RiboBio Co., Ltd.) was used to detect proliferation rate of NCIH1650 and A549 cells. After transfection of 24 h, 8,000 cells/100 µl were plated in 96-well plates overnight and then incubated with 100 µl 50 µM EdU solution for 2 h. The cells were then fixed with 50 µl 4% paraformaldehyde at room temperature for 10 min and neutralized using 50 µl 2 mg/ml glycine. After permeabilization with 0.5% Triton X-100 for 20 min, the cells were stained for DAPI with 100 µl 1× Apollo solution and stained for Edu with 100 µl 1× Hoechst33342 solution, both at room temperature for 30 min. Images were captured using fluorescence microscopy with 40 times magnification and proliferation cells ratios were counted from three random fields of view.

A total of 1.5×10⁵ transfected cells were plated into 6-well plates, and the assay performed according to the manufacturers protocol of a Cell-Light™ EdU Apollo^® 488 In Vitro Imaging kit (C10310-3; Guangzhou RiboBio, Co., Ltd., Guangzhou, China). Finally, cells were visualized and counted using a fluorescence microscope (Leica Microsystems, Wetzlar, German).

Cell proliferation assays were performed 24 h after transfection. For CCK8 viability assay, cells were seeded at a density of 2,000 cells/well in a 96-well plate. 10% CCK8 (Beyotime, Suzhou, China) was added to the well, and the absorbance at 450 nm was measured every 24 h. For EdU assay, 8,000 cells/100 μL were plated in 96-well plates. One hundred microliters of 50 μM EdU solution (EdU Apollo488 In Vitro Imaging Kit, RiboBio, Guangzhou, China) was incubated with cells. EdU was stained with red fluorescence, and images were photographed by fluorescence microscope to count the proportion of proliferating cells.

LUAD cells, A549 and PC9, were transfected with plasmids or small interfering RNAs (siRNAs) prior to cell proliferation assays. For the EDU (5-ethynyl-2′-deoxyuridine) assay (EdU Apollo^®488 In Vitro Imaging Kit, RiboBio, Guangzhou, China), 15◊103 cells/100 µL/well were seeded into a 96-well plate. Once the cells adhered to the plate, 100 µL reagent A was added in the wells and allowed to incubate at 37 ºC for 2 h. Afterward, the cells were fixed with 4% paraformaldehyde (Biosharp), treated with 0.5% TritonX-100 (100 µL), then subjected to Apollo and Hoechst staining. After washing with phosphate-buffered saline (PBS) three times, the cells were photographed using a fluorescence microscope to calculate the proliferation rates. In CCK-8 assay, 2◊10³ cells/well were seeded into 96-well plates and 10 µL CCK-8 reagent was added. And then the plates were incubated for 2 h under 5% CO₂ environment. After 0-, 24-, 48-, 72-, and 96-h incubation, absorbance at 450 nm was recorded. To evaluate colony-formation, 400 cells/well were seeded into 6-well plate and cultivated for 10–14 days. Following incubation, the cells were fixed in 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet solution, washed with PBS, and then photographed.