Cck 8 reagent

The Cell Counting Kit-8 (CCK-8) test kits (Cat#HY-K0301, MedChemExpress, Shanghai, China) was utilized in order to determine the cellular proliferation of H1299 and H226 cells with overexpressing GPD1L. The cells were firstly inserted onto 96-well plates (2500 cells per well, 100 μl) after GPD1L-overexpressing plasmids were transfected. Afterwards, the cells were applied for CCK-8 assays at 24, 48, 72, and 96 hours. After adding 10 μl of CCK-8 reagents from MedChemExpress company (Cat#HY-K0301, Shanghai, China) and allowing the mixture to incubate for one hour, the optical density was measured using a microplate reader from BioTek (BioTek, Winooski, VT, USA) at a wavelength of 450 nm.

HIEC-6 cells (5 × 10³ cells per well) were plated into 96-well plates and incubated for 24 h, and then, different concentrations of Sitagliptin were added into medium. After another 24 h treatment, CCK-8 reagents (MedChemExpress, USA) were added to assess cell viability using absorbance detected by a microplate reader (BioTek, USA) at 450 nm wavelength.

After transfection, MDA-MB-231 and MCF-7 cells were inoculated in 96-well plates with 2000 cells per well and cultured for 24 h. According to the manufacturer's instruction, 10 μL of CCK-8 reagents (Med Chem Express, Monmouth Junction, NJ, USA) were added to each well at the 24th h, 48th h and 72nd h, and then the cells were incubated for an additional 1 h. The absorbance (OD) values of each well at 450 nm wavelength were detected at each indicated time point by a microplate reader (Bio-Rad, Hercules, CA, USA).

To evaluate the proliferation of T lymphocytes, whole blood (10 mL) was collected from immunized piglets in each group at 42 dpi, and peripheral blood lymphocytes were isolated using a pig peripheral blood lymphocyte isolation kit (TBDsciences, Tianjin, China) in accordance with the manufacturer’s protocol. The lymphocyte proliferation assay was performed as described in our previous study [14 (link)]. Briefly, lymphocytes (4 × 10⁶ cells/mL) were seeded into a 96-well plate with 100 µL RPMI-1640 containing 20% FBS and stimulated with purified APPV E2 protein (10 µg/mL), concanavalin A (10 µg/mL), and 100 µL RPMI-1640 containing 20% FBS. After 72 h of culture at 37 °C, 10 µL CCK-8 reagent (MedChemExpress, Shanghai, China) was added to each well and incubated at 37 °C for 4 h. Finally, the absorbance was measured at 450 nm wavelength, and the stimulation index (SI) was calculated according to the formula: SI = (OD values of immunized groups − OD values of blank control)/(OD values of negative control − OD values of blank control).