Multiskan mk3 plate reader

The in vitro viability and cytotoxicity were determined by CCK-8 assay and protocols (Beyotime). In viability assay, after A549 and A549/PEM LUAD cells were subjected to the relevant transfection or/and PEM exposure, CCK-8 reagent was added to the cells, followed by incubation for 2 h at 37 ℃. In cytotoxicity assay, un-transfected or transfected LUAD cells were challenged with various concentrations of PEM for 24 h. After that, CCK-8 reagent was used as described. Plates were read using a Thermo MULTISKAN MK3 plate reader at 450 nm. Based on the plot of the percentage of viable cells relative to PEM concentration, we scored the IC50 value.

All immunized mouse serum samples were heat-inactivated at 56 °C for 30 min before use. Briefly, 96-well plates (Beijing Wantai Biological Pharmacy, Beijing, China) were coated overnight at 4 °C with 0.5 µg/well of purified SARS-CoV-2, BANAL-20-52, or BANAL-20-236 RBD proteins in PBS. After blocking with 5% fat-free dry milk in TBS-T (blocking buffer) for 1 h at room temperature, 5-fold serially diluted heat-inactivated sera (starting dilution 1:100) in blocking buffer were added to the plates. After 1 h incubation, the plates were washed three times, and horseradish peroxidase HRP-conjugated goat anti-mouse IgG in blocking buffer at a dilution of 1:5000 was added to the plates and incubated at room temperature for 1 h. After washing three times, 100 μL of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Beijing Wantai) was added into each well and incubated in the dark at room temperature for 15 min. The reaction was then stopped with 50 μL of stop solution (Beijing Wantai), and the absorbance at 450 was recorded using a MultiSkan MK3 plate reader (Thermo, Rochester, NY, USA). The IgG endpoint GMTs were defined as the dilution fold, which emitted an optical density exceeding 2× background (without serum but the secondary antibody was added).

Dipeptidyl-peptidase IV (DPP-IV) inhibitory activity was assessed according to the method described by Zeng et al. (12 (link)) with some modifications. Briefly, 25 μL Gly-Pro-p-nitroanilide (6 mM; Sigma-Aldrich) and 25 μL Tartary buckwheat sample, or 25 μL phosphate buffer saline (PBS; Sigma-Aldrich) as a control, were mixed and preincubated at 37 °C for 10 min. The reaction was initiated by adding 50 μL DPP-IV from porcine kidney (3·10^–4 U/L, ≥10 U/mg protein; Sigma-Aldrich) and the mixture was incubated at 37 °C for 60 min. The reaction was terminated by adding 100 μL of 1 M sodium acetate buffer (pH=4.0; Sinopharm Chemical Reagent Co., Ltd), and the absorbance of the samples at 405 nm was measured on a Multiskan MK3 plate reader (Thermo Fisher Scientific). Each sample was analyzed in technical triplicate, and the absorbance values were normalized to sample blanks in which DPP--IV was replaced with Tris-HCl buffer (0.1 M, pH=8.0; Solarbio, Beijing, PR China). The negative control (no DPP-IV activity) and positive control (DPP-IV activity with no inhibitor) were prepared by using Tris-HCl buffer (100 mM, pH=8.0; Solarbio) instead of the sample or instead of the DPP-IV solution and the sample, respectively. Diprotin A (Sigma-Aldrich) was used as a standard inhibitor. The DPP-IV inhibition rate was calculated using Eq. 3.