Emax endpoint elisa microplate reader

Cells were seeded 96-well plates with time-course incubations, and were then washed with phosphate-buffered saline (PBS) with MTT (0.5 mg/mL PBS) and incubated at 37 °C for 30 min. Formazan crystals were dissolved with dimethyl sulfoxide (40 μL/well) and detected at OD₅₇₀ using an Emax Endpoint ELISA microplate reader (Molecular Devices).

Collagen fibrillogenesis was monitored at 37 °C by a temperature-controlled microplate spectrophotometer (EMax End point ELISA microplate reader, Molecular Devices, Sunnyvale, CA). The collagen (type 1) from the rat tail was dispersed in acetic acid (20 mM) at a concentration of 3 mg mL⁻¹ and diluted in HEPES (0.2 M) and 1.0 M PBS (pH 7.4) in an 8:1:1 volume ratio. The collagen solution was neutralized on ice and transferred into the 96-well microplate. Each 50 μL of the control (DI water), RB (500 μM), HA-RB conjugates (500 μM), ZnS:Ag,Co particles, and HA-RB/ZnS:Ag,Co mixtures was mixed with 100 μL of the collagen solution (n = 3). The absorbance from 350 to 370 nm was acquired at 1 min intervals for 6 min under UV light. During irradiation, the light was turned on and off every 30 s (On/Off cycle: 30 s/30 s). The group of HA-RB conjugates with green light was monitored separately under the same conditions.

The levels of HB-EGF secretion in the cell supernatants or mouse plasma were analyzed by factor-specific ELISA according to the manufacturer’s protocol (R&D Systems, MN, USA). The absorbance was measured at 450 nm using an EMax Endpoint ELISA microplate reader (Molecular Devices Corporation, CA, USA).

A diphenyltetrazolium bromide (MTT) assay was performed to determine the cell viability, as measured by the metabolic activity of cells. A total of 1 × 10⁴ cells were seeded in each well of a 96-well plate and cultured overnight before treatment. After treatment, the culture medium was aspirated, and 0.5 mg/mL MTT solution (Sigma-Aldrich, Saint Louis, MO, USA) was added to each well before incubation for 3 h at 37 °C. The supernatant was aspirated, and dimethyl sulfoxide (Sigma-Aldrich) was added to release and form acriflavine. The resulting absorbance was measured at 540 nm using the EMax^® (Endpoint ELISA Microplate Reader; Molecular Devices Inc., San Jose, CA, USA).

The concentrations of VEGF, PDGF-BB, interleukin- (IL-) 10 (R&D Systems, Minneapolis, MN, USA), and TNF-α (BioLegend Inc., San Diego, CA, USA) in serum and BM were measured using ELISA according to the manufacturer's instructions. Optical density was measured at 450 nm using an EMax Endpoint ELISA Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

The amount of NO in the CMEC-conditioned medium was quantified using the Griess reagent system, and the absorbance was measured at 540 nm using an EMax Endpoint ELISA Microplate Reader (Molecular Devices). The amount of nitrite in the culture media was calculated using sodium nitrite as a reference standard.