Cck 8 assay

The cytotoxicities of TGC, ERC, OMC, and SAC in the THP-1 macrophage cell line were determined by cell counting kit-8 (CCK-8) assay (Solarbio life sciences, China). The THP-1 cells were purchased from Wuhan Procell Life Science & Technology Co., Ltd., and cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) (lot no. 2393124RP; Gibco, United States) at 37°C and 5% CO₂. Cells were plated into a 96-well plate at a density of 3 × 10⁴ cells per well and incubated with 200 ng/mL phorbol myristate acetate (PMA). The macrophages were differentiated over 48 h, the PMA removed, and the cells washed twice and finally treated with the four tetracyclines at different concentrations (5 mg/L, 10 mg/L, 20 mg/L, and 40 mg/L) in RPMI 1640 supplemented with 5% FBS. After incubation for another 24 h, 100 μL of medium containing 10% CCK-8 solution was added to each well and the culture maintained for an additional 2 h. Finally, a Multiskan FC microplate reader (Thermo Fisher, USA) was used to measure the absorbance at 450 nm. According to the kit’s instructions, cell viability in drug-treated groups was defined as the percentage of the value for control group cells after eliminating the impact of background absorbance.

CCK-8 assay (Solarbio, Beijing, China) was used to determine the effect of Rg1 on the cell viability of NP cells. For the detection of ginsenoside Rg1 and IL-1β cytotoxicity, the NP cells with or without IL-1β induced were seeded in a 96-well plate (5 × 10³/well) with three replicate wells per group and intervened by various concentrations of ginsenoside Rg1 (0, 10, 20, 50, 100, 200 μmol/L). After being cultured for 24 h, the cells in each well were incubated with CCK-8 solutions for 4 h at 37℃. Then, the absorbance was measured at 450 nm on an automatic microplate reader (ELX80, Bio-Tek, Winooski, VT). As for the cell proliferation, the cells were seeded in a 96-well plate (2 × 10⁴/well) with three replicate wells per group and cultured for 24 h, 48 h, and 72 h. Then, 10 μL of the CCK-8 solution was added in each well, and incubated at 37℃ for 4 h. The absorbance was checked as above. The experiment was repeated three times. And the proliferation curve was plotted with time point as the X-axis and A value as the Y-axis.

Cell proliferation was evaluated using a CCK8 assay (Solarbio, Beijing, China). Cells were quantified, and 1,000 cells were dispensed into 96-well plates, each well containing 100 μl of medium. They were then treated with CD27 antibody (5 μg/ml, Varlilumab, 1F5, Chemstan, Wuhan, China) and Soluble CD27 (sCD27, 1 ng/ml, ab114342, Abcam, UK) at intervals of 0 h, 48 h, and 72 h. A dimethyl sulfoxide (DMSO, 5ul/ml) treated group served as the control. Subsequently, 10 μl of CCK-8 reagent was introduced to each well, followed by an additional 0.5 h of incubation at 37℃ in a cell incubator (Thermo Forma 311, USA). The absorbance at 450 nm was then ascertained using a microplate reader.
For apoptosis assessment, cells were harvested to yield single-cell suspensions. After undergoing two washes with PBS as the staining buffer, the cells were incubated in the dark at 4℃ for 30 min. They were then stained with Annexin V-FITC and propidium iodide (PI) for 15 min, after which apoptosis was gauged via flow cytometry.

293T, H1299, and Caco-2 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco). A549 cells were grown in Kaighn’s Modification of Ham’s F-12 medium (F-12K, Gibco). All media were supplemented with 10% fetal bovine serum (Gibco), 100 units/mL penicillin, and 100 µg/mL streptomycin, and cells were grown at 37°C under an atmosphere of 5% CO₂. The H1299 cells used for trVLP or live SARS-CoV-2 infection studies were infected with lenti-ACE2, and stable clones were selected by screening with puromycin (1 µg/mL). Cells were treated with H89 (Selleck), 666-15 (Selleck), and remdesivir (Selleck). The cytotoxicity of the drugs used in this study was tested by a CCK-8 assay (Solarbio). Transient transfection was performed with Lipofectamine 2000, Lipofectamine 3000 (Invitrogen), or the TransIT-X2 dynamic delivery system (Mirus) according to the manufacturer’s instructions.

Cell proliferation was assessed using the CCK-8 assay (Beijing Solarbio Science & Technology Co., Ltd.). Transfected cells were treated and cultured in 96-well plates at 5 × 10³ cells/well, followed by incubation with CCK-8 solution (10 µL) in accordance with the manufacturer’s instructions. The optical density was measured at 450 nm using an ultraviolet spectrophotometer (Agilent Technologies, USA).