Microplate spectrophotometer

The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay was used to determine alveolar macrophage viability. At least (5 × 10⁴ cells/well) were plated in 96-well plates and allowed to adhere for 3 h. Then, DMEM was replaced with fresh medium and cells were treated with increasing concentration of umPEA (10⁻⁹–10⁻⁵ μM). MTT stock powder was obtained by Sigma-Aldrich (Milan, Italy). After 24 h, 25 μL MTT (5 mg/mL MTT in DMEM) was added to the cells, and the mixture was incubated for further 3 h at 37 °C. Subsequently, the cells were lysed and the dark blue crystals were solubilized using a 100 μL solution containing 50% N,N-dimethylformamide and 20% (w/v) sodium dodecyl sulphate (SDS) (pH 4.5). The optical density (OD) of each well was determined using a microplate spectrophotometer equipped with a 450 nm filter (PerkinElmer, Inc.; Waltham, MA, USA).

Serum samples collected from 8-week-old female Tlr7^−/− NOD mice and Tlr7^+/+ NOD mice were tested for anti-insulin autoantibodies by ELISA. Plates were coated with human insulin (4 μg/ml, Lilly) overnight. After washing and blocking (1 h, room temperature, 1% BSA in PBS), diluted (1:100) serum samples were tested for total (Ig) anti-insulin autoantibodies and different isotypes of anti-insulin autoantibodies with Alkaline phosphatase-conjugated (AP-conjugated) goat anti-mouse IgH+L, IgM, IgG, IgA, IgG1, IgG2a, IgG2b, IgG3 and a phosphate substrate. The enzymatic reaction was stopped with NaOH, and the plates were read with a microplate spectrophotometer (Perkin Elmer, Waltham, MA, USA) at OD 405 nm. Different isotypes of total serum immunoglobulins were also measured. Briefly, the wells of a 96-well plate were coated with samples or standards overnight. After washing and blocking (1 h, room temperature, 1% BSA in PBS), the plates were then incubated with AP-conjugated goat anti-mouse IgG1, IgG2a, or IgG2b (2 h, room temperature). The samples were subsequently washed, and the substrate PNPP (Sigma) was added. The reaction was stopped by adding 1 M NaOH, and the samples were analyzed on a microplate spectrophotometer at 405 nm (OD).

Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8, GLPBIO) assay according to the manufacturer’s instructions. Briefly, ESCC cells were seeded in triplicate in 96-well plates with a density of 2,000 cells per well, and infected with P. gingivalis and transfected with Beclin1 siRNA under the conditions described above. Then, 100 μl culture medium was added to each well, and the cells were cultured in an incubator with 5% carbon dioxide at 37°C for 24 h. After fresh culture medium was changed, 10 μl of CCK-8 reagent was added to each experimental well, followed by incubation under the above culture conditions and the optical density (OD) value was measured using a microplate spectrophotometer (PerkinElmer, Waltham, MA, United States) at 490 nm. The cell viability rate was calculated by the discrepancy between the OD value of the experimental well and the control well after removing the OD value of the blank well. The experimental wells were mixed with infected P. gingivalis or transfected with Beclin1 siRNA ESCC cells, cell culture medium and CCK-8 reagent; The control wells were mixed with wild type ESCC cells, cell culture medium and CCK-8 reagent; The blank wells were mixed with cell culture medium and CCK-8 reagent; The experiments were performed in triplicate with three parallel samples for each and the representative results are shown.

SARS-CoV-2 WT, Omicron BA.1.1.529, BA.2.12.1, BA.2.13, and BA.4/BA.5 spike plasmids were constructed using the pcDNA3.1 vector. G*ΔG-VSV virus (VSV G pseudotyped virus) was used to infect 293T cells, and spike protein-expressing plasmid was used for transfection at the same time. After culture, the supernatant containing pseudovirus was collected, filtered, aliquoted, and frozen at −80 °C for further use. Monoclonal antibodies were serially diluted (threefold) in DMEM (GIBCO, USA) and mixed with pseudovirus in 96-well plates. After incubation at 5% CO2 and 37 °C for 1 h, digested Huh-7 cells were seeded. After 24 h of culture, supernatant was discarded and d-luciferin reagent (PerkinElmer, USA) was added to react in the dark. The luminescence value was measured using a microplate spectrophotometer (PerkinElmer, USA). IC50 was calculated by a four-parameter logistic regression model using PRISM v 8.0.1.

After seven days of hypoxia, blood samples in each group were collected to separate the serum and blood after received 80 mg/Kg acipimox. Fasting serum levels of free fatty acids (FFAs)in each rat were assessed using the corresponding commercial kits (MEIMIAN, Guangzhou, China). After processing the samples according to the instructions, we measured the absorbance at 450 nm using a microplate spectrophotometer (PerkinElmer, Waltham, MA, USA), and calculated the FFA content.