Automatic microplate reader

Anthropometric measurements and blood analysis were conducted at baseline and at study completion. Heights and bodyweights were measured using an anthropometer and a scale. Body mass indices (BMIs, kg/m²) were calculated by dividing weight by height squared. Blood was collected from subjects in a 10-h fasted state. Inflammatory factors in serum, that is, interleukin (IL)-4, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and monocyte chemotactic protein (MCP)-1, were automatically analyzed using Quantikine ELISA Human lmmunossay kits (R&D Systems Inc., MN, USA) and an automatic microplate reader (Molecular Devices, USA) at the Seegene Medical Foundation (Seoul, South Korea).

Either the full-length of circINSR or three target-site fragments were inserted into the psiCHECK-2 vector (Promega, Fitchburg, WI, USA). A miR-34a biosensor (psiCHECK-2-miR-34a 2×) was created by inserting two copies of miR-34a reverse complementary sequence in the psiCHECK-2 vector. The predicted 3′ UTR fragment containing the miR-34a binding site in CCNE2 and Bcl-2 was PCR amplified, cloned into the psiCHECK-2 vector, and designated gene-WT. To mutate the presumptive binding sites in CCNE2 and Bcl-2, the binding site sequences were replaced as indicated, and designated gene-MUT.
For the luciferase reporter assays, HEK293T cells were seeded in 96-well plates at 8 × 10³ cells per well and co-transfected with 0.2 μg reporter plasmid and 50 nM miR-34a mimic or mimic-NC. The ratio of firefly and renilla luciferase activity was detected with the dual-luciferase reporter assay kit (E2920, Promega, Fitchburg, WI, USA) after 24 h. The optical density of the resulting solution was assessed using the automatic microplate reader (Molecular Devices, Sunnyvale, CA, USA).

The tests were conducted according to our previous reports [36 (link),37 (link)]. The cells were grown in DMEM medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) in a 37 °C and 5% CO₂ incubator. The cytotoxic activities were performed by the MTS assay. Briefly, 28 compounds were dissolved with DMSO. The cells were seeded at 1 × 10⁵ cells/mL in 96-well plates (100 µL/well) and cultured for 18 h when the cells filled the bottom of the wells. Then the supernatant was discard, the compounds (10 μM, 100 µL/well) were added to the culture and incubated for 24 h before cell viability measurement. 10 µL MTS reagent (0.5 mg/mL) were added and further incubated for 4 h. Finally, absorbance was read using an automatic micro-plate reader (Molecular Devices, USA) at 490 nm to test the cell viabilities, and the results were presented as the percent of non-treated control for each concentration. Irinotecan was used as the positive control. All measurements were repeated in triplicate. Data were expressed as the mean ± SD.

HEK-293 T cells were plated in 96-well plates for 24 h before transfection. The cells were co-transfected with psi-CHECK-2 reporter plasmid, miR-27a-3p mimics, si-RNA or pCD2.1-circRNF111 vector. Cells were harvested 24 h after transfection. The ratio of Renilla and Firefly luciferase activity was detected with the Dual-Luciferase Reporter Assay Kit (#E2920, Promega, Fitchburg, WI, USA). The optical density of the resulting solution was assessed using the automatic microplate reader (Molecular Devices, Sunnyvale, CA, USA).

HEK293T cells were seeded in 96-well plates for 24 h before transfection. The cells were co-transfected with psiCHECK-2 reporter plasmid, miR-145 mimics, siRNA, or PCD2.1-circRILPL1 vector. The ratio of renilla and firefly luciferase activity was detected with the Dual-Luciferase Reporter Assay Kit (#E2920, Promega, Fitchburg, WI, USA) after 24 h. The optical density of the resulting solution was assessed using the automatic microplate reader (Molecular Devices, Sunnyvale, CA, USA).