Cck 8

Huh7 and PLC/PRF/5 cells stably infected with lentivirus (2×10³/well) were seeded in 96-well plates and incubated at 37°C overnight. These HCC cells were then incubated at 37°C for 24, 48, 72 or 96 h. CCK-8 (Dojindo Molecular Technologies, Inc.) was used to determine the viability of proliferating cells, according to the manufacturer's protocol. Briefly, HCC cells were incubated at 37°C for 2 h after adding 10 µl/well CCK8 reagent, and the absorbance was measured at 450 nm using an Infinite M200 Pro Multifunctional microplate reader (Tecan Group Ltd).

Referring to the protocol of the CCK-8 (cat. no. C0038; Beyotime Institute of Biotechnology), NCM460, SW480, SW620, LOVO, HT29 and HCT116 cells (2×10⁴ cells/well) were inoculated into 96-well plates. Afterwards, 20 µl CCK-8 solution and 100 µl DMEM (with 10% FBS) were added and cells were incubated for 1 h. The absorbance values at 450 nm were measured by a microplate reader (Tecan Group, Ltd.).

Hsa_circ_0137606-wild type (WT) and hsa_circ_0137606-mutant (Mut) were constructed using pGL3-Basic luciferase vectors (Promega Corporation) and transfected into BC cells with or without NC or miR-1231 mimics, respectively. After 48 h, a dual-luciferase reporter assay kit (Promega Corporation) was used to determine lucif-erase activity. Renilla luciferase was used for normalization.
The luciferase activities were measured using a luciferase assay kit (Promega Corporation). Three independent experiments were performed in triplicate.
Cell Counting Kit-8 (CCK-8) proliferation assay. Transfected cells were cultured in 96-well plates (1,000 cells/well) for 0, 24, 48, 72 or 96 h. According to the manufacturer's protocols, a CCK-8 (Dojindo Molecular Technologies, Inc.) was used to detect cell proliferation. A total of 10 μl of CCK-8 solution was added to each well of the 96-well plate and incubated at 37°C for 2 h. The absorbance (450 nm) was measured using a Sunrise Microplate Reader (Tecan Group, Ltd.). Three independent experiments were performed in triplicate.

The CCK-8 (Sangon Biotech Co., Ltd.) was used to determine the viability of U251 and U87 cells. Transfected cells (2×10³/well) in 96-well plates were incubated for 0, 24, 48 and 72 h. Then, 10 µl CCK-8 reagent was added to each well and the cells were incubated for a further 2 h. The absorbance was measured at an optical density of 450 nm using a multimode microplate reader (Tecan Group, Ltd.).

Cell viability was assessed using a CCK-8 (Dojindo Molecular Technologies, Inc.), according to the manufacturer's instructions. Cells (3×10³/well) were seeded into 96-well plates and cultured overnight. At 0, 24, 48 and 72 h, the CCK-8 reagent (10 µl) was added to each well and incubated at 37°C for 1 h. The absorbance was then measured at 450 nm (optical density value) using an automated microplate reader (Tecan Group, Ltd.) Each experiment was performed ≥3 times independently.