Cck 8 solution

Cell proliferation was assessed using the CCK8 assay and colony formation assay. For the CCK8 assay, after cell transfection, 1 × 10³ GBM cells were seeded on 96-well plates. After 24, 48, and 72 hours of incubation, CCK-8 solution (APExBIO, USA) was added to each well for further incubation of the cells and the absorbance at 450 nm was measured with a spectrophotometer. For the colony formation assay, after cell transfection, cells were seeded in 6-well plates at a density of 500 cells/well and cultured for 2 weeks. After the colonies had formed, they were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted as part of clonogenic assays.

Lymphocytes were isolated from specific-pathogen-free mouse spleens using the Spleen Lymphocyte Isolation Kit (Solebol Reagent Co., Ltd., Beijing, China). The cells were seeded in 96-well plates at a concentration of 3 × 10⁵ cells per well and stimulated with 10 μg/mL stimulatory antigen (purified recombinant proteins VP2-VLP, L₂B, NT₁L₂B, NT₁L₂B₄B, or N(T₁)₂L₂B₄B, or PPV AV30 viral particles). After incubation for 72 h, CCK-8 solution (APExBIO, Houston, TX, USA) was added and the cells were incubated for 4 h. The OD₄₉₀ values were determined with a microplate reader. Concanavalin A (ConA, 5 μg/mL) and RPMI-1640 (Thermo Fisher Scientific Inc., USA) were used as the positive and negative controls, respectively, with six replicates in each group.

After transfection and treatment, AC16 cells were collected and then re-seeded into 96 well plates. Then, 48 h later, the cells were incubated with cell counting kit 8 (CCK8) solution (Apexbio, Houston, TX, United States) for 4 h. The absorbance at 450 nm was observed under a microplate reader (Bio-Rad, Hercules, CA, United States).

Cell suspension was inoculated in 96-well plates, and CCK8 solution (APExBIO, USA) was added to each well. After incubation, absorbance was measured at 450nm with a microplate reader, and then cell proliferation inhibition rate was calculated. All samples were performed in triplicate.

Briefly, different diluted antisera (anti-IDE and anti-SVA pig sera) and normal guinea pig serum as controls were used in IBRS-2 cells to perform the neutralization test. Fifty microliters of the antiserum treated at 56°C for 30 min was serially diluted 2-fold from 1:4 to 1:512. Then, the diluted antisera were mixed with SVA strain CH-FJ-2017 (200 TCID₅₀) and incubated for 1 h at 37°C in a 96-well plate. Subsequently, 100 μL of the mixtures was transferred to IBRS-2 cells in a 96-well plate and incubated for 1 h at 37°C. The cell supernatant was discarded, and after three washes, DMEM containing 2% FBS was added. After incubation for 48 h, CCK-8 solution (APExBIO, USA) was added to the cells, and cell viability was detected according to the instructions of the CCK-8 assay kit. The percent protection from CPE was measured as described by Li et al. (49 (link)) with minor modifications. The protection from CPE (%) was calculated as follows: 100 × (OD 450 of each serum – OD 450 of PC)/(OD 450 of cell control – OD 450 of PC), with Positive control (PC) representing a virus infection control. Each serum sample was assayed in triplicate, and the data are presented as the average value of triplicate results.

OSCC cells (5,000 cells in 100 μL medium per well) after 48 h of transfection were seeded into 96-well plates. After incubation for 0, 24, 48, and 72 h, 10 μL of CCK8 solution (Cat#: K1018; APExBIO, China) was added to the wells and incubated for another 4 h. The optical density (OD) values at 450 nm were measured using a multimode plate reader (Thermo, USA) to assess cell proliferation according to a previous study [30 ].