Automated elisa reader

Human ASM cells were cultured in six-well plates with Dulbecco's modified Eagle medium (DMEM), supplemented with L-glutamine, 10% newborn calf serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin in a 5% CO₂ atmosphere at 37°C for 24 h as previously described [17] (link), [18] (link). Next, the cells were trypsinized and diluted to a concentration of 1×10³ cells/100 µL in serum free medium. A total of 100 µL human ASM cells were added to each well of 96-well plates. After incubating overnight, the cells were treated with recombinant human CTGF at a final concentration of 0–30 ng/mL and cultured for 1–5 days. For the proliferation inhibition assay, the cells were incubated in 10 ng/mL CTGF and 100 ng/mL anti-CTGF scFv monomer, 100 ng/mL anti-CTGF scFv dimer, or 1 µg/mL LY294002 for 1–5 days. A total of 20 µL MTT reagent [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL] was added to each well, and the cells were incubated at 37°C for 4 h. The medium in each well was then replaced with 150 µL dimethylsulfoxide (DMSO). The plates were incubated on a shaker for 15 min at room temperature, and then the absorbance was measured using an automated ELISA reader (Bio-Rad, USA) at 490 nm.

MTT [3‐(4, 5‐dimethylthiazol‐2‐yl)‐ 2,5‐ diphenyltetrazolium bromide] assay (Sigma, St. Louis, MO) was used to examine cell proliferation. Briefly, at different times (1–7 days) posttransfection, MTT (0.25 mg/mL) was added to each well and the plates were incubated for 4 h at 37°C and 5% CO². After removing the MTT solution, 180‐μL dimethyl sulfoxide (DMSO) was added and the plates were incubated for 15 min at 37°C to dissolve the formazan crystals. The absorbance values were measured at 570 nm using an automated ELISA reader (Bio‐Rad, Hercules, CA). Alternatively, cell viability was evaluated with the trypan blue staining assay.

To measure glutamate concentration, spinal cord and serum samples from control, carrageenan-injected, and capsazepine-treated animals were collected 1 h after intraplantar carrageenan injections, the timepoint at which the anti-nociceptive effect of capsazepine reached its peak. Glutamate concentrations were measured using a colorimetric glutamate assay kit (Abcam, Cambridge, UK), which identifies glutamate as a specific substrate and forms a color product at 450 nm. Spinal cord and serum samples were homogenized in RIPA buffer containing protease and phosphatase inhibitors. Homogenates were centrifuged for 20 min at 10,000× g at 4 °C to remove solid material, and the supernatants were transferred to new tubes. Supernatant aliquots were used for quantitative measurements of glutamate concentration. An automated ELISA reader (Bio-Rad Laboratories, Hercules, CA, USA) was used to read the color of the reaction.