Softmax

HAECs were plated in 24-well culture plates at a density of 5 × 10⁴ cells/well and cultured overnight. Culture media were replaced with 250 μL of EGM-2 containing test drugs and incubated for 1 h. Supernatants were collected and stored at –80°C until further analysis. PGI₂ was assessed as 6-keto prostaglandin F_1α (6-keto PGF_1α) using the 6-keto PGF_1α enzyme immunoassay (EIA) kit (Cayman Chemical, Michigan, USA) according to the manufacturer’s instructions. Optical density was measured at 405 nm using a microplate reader (Soft max, Molecular Devices, Sunnyvale, CA, USA). Results are expressed as 6-keto PGF_1α concentration (pg/mL).

Specific serum IgG titers were measured by enzyme-linked immunosorbent assay (ELISA) as described previously^{9 (link),48 (link)}, using sonicated whole cell extracts of each periodontal bacteria. Briefly, 96-well microplates (EIA plate; Costar, Corning, NY, USA) were coated with sonicated A. actinomycetemcomitans, F. nucleatum, or P. gingivalis at 10 μg/mL in carbonate buffer and incubated for 2 h at 37 °C. After blocking with 2% bovine serum albumin in carbonate buffer, plates were washed with phosphate-buffered saline (PBS)-Tween (1×PBS, 0.05% Tween 20, pH 7.2). Serially diluted reference positive control serum (2⁵–2¹⁵, 200 µL per well) and single diluted patient serum (2¹⁰, 200 µL per well) were added into each well, after which the plates were incubated for 1 h at 37 °C and washed again. Subsequently, 200 µL per well of alkaline phosphatase-conjugated goat anti-human IgG (Sigma, St. Louis, MO, USA) was added. Following incubation, plates were washed and developed with phosphatase substrate (Sigma). Optical density at 450 nm was read using a microplate reader (SoftMAX, Molecular Devices, Sunnyvale, CA, USA). Antibody titers were calculated according to the method of Wang et al.^{48 (link)}.

HAECs were plated in 96-well culture plates at a density of 1 × 10⁴ cells/well and cultured overnight. Culture media were replaced with 250 μL of EGM-2 containing test drugs and incubated for 1 h. Supernatants were collected and stored at –80°C until further analysis. IP3 was assessed using human inositol 1,4,5-trisphosphate, IP3 ELISA Kit (Cusabio, Wuhan, China) according to the manufacturer’s instructions. Optical density was measured at 450 nm using a microplate reader (Soft max, Molecular Devices). Results are expressed as IP3 concentration (pg/mL).

ELISA was used to measure the concentration of IL-1β (eBioscience), TNF-α, CXCL1 (R&D systems DuoSet), IL-6^{40 (link)} and IFN-α^{40 (link)}. Absorbance was determined at 450 nm, on SpectraMax Plus (Molecular Devices) or FLUOstar Omega (BMG Labtech) plate readers and analyzed using SoftMax (Molecular Devices) or Mars (BMG Labtech) software.

IL-10, TGF-β, IL-6, IL-17, and IL-23 cytokine levels were tested in the plasma of patients and healthy subjects utilizing DuoSet sandwich enzyme-linked immunosorbent assay (ELISA) development kits (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's guidelines. The intensity of the formed color was assessed using a microplate reader (Sunrise; Tecan Group Ltd, Männedorf, Switzerland) to read optical absorbance at 450 nm. The digital data of raw absorbance values were processed into a standard curve by the ELISA reader-controlling program (Softmax; Molecular Devices, Sunnyvale, CA, USA), from which the cytokine concentrations of the specimen were calculated. Results were expressed as a picogram of cytokine per milliliter plasma (pg/ml).

Sheep red blood cells (RBCs) were opsonized with a subagglutinating concentration of IgG rabbit antisheep erythrocyte antibody (Cappel Organon Teknika, Durham, NC) as previously described [29 (link)]. The phagocytosis of IgG-coated RBCs (IgG-RBCs) was assayed using adherent macrophage monolayers in DMEM medium as described previously [30 (link)]. Briefly, rat AMs or mouse PMs or BMDM were cultured in 96-well plates as described and preincubated with TLR agonists for 5–60 min. Following preincubation, opsonized RBCs were added at a target: cell ratio of 40 : 1, and cultures were incubated for an additional 90 min at 37°C. Wells were then washed three times with PBS to remove uningested erythrocytes. Serial dilutions of known amounts of RBCs were added to separate wells to derive a standard curve, and 75 μl of 0.3% SDS in PBS was added to each well for 10 min. Lastly, 50 μl of 3,3′,5,5′-tetramethyl-benzidine dihydrochloride hydrate (TMB) (0,2 mg·ml⁻¹) solution was added to each well as a chromogen. Following a 5 min incubation (at room temperature) in the dark, the reaction was stopped with 100 μl H₂SO₄ (2 M) and the absorbance was evaluated at 450 nm with an automated reader (SOFTmax, Molecular Devices). The number of RBCs per well was derived from absorbance data at 450 nm using the standard curve made. Independent experiments were performed in septuplet.