Tryptase

The human mast cell line was purchased from Kerafast (Kerafast, MA, USA) and maintained in StemPro-34 SFM at a concentration of 5 × 10 5 cells/ml. The cells were grown at 37 °C with 5% CO 2 , and the medium was exchanged weekly with 50% fresh medium without exogenous cytokines. The mast cells were seeded in 6-well plates at a density of 5 × 10 4 cells/ml. After one day, the medium was replaced with StemPro-34 SFM culture medium with or without cytokines. The mast cells were then stimulated with 100 ng/mL human recombinant IL-33 (R&D Systems, Inc.) for 24 and 72 h. The supernatants and RNA were harvested and stored at - 80 °C until analysis.
Enzyme-linked Immunosorbent Assay (ELISA) SF and serum samples obtained from healthy volunteers and patients with RA and osteoarthritis were subjected to tryptase, chymase, and histamine analyses using tryptase (Boster Biological Technology, CA), chymase (Aviva Systems Biology, CA), and histamine (Enzo Life Sciences Inc., NY) ELISA kits, according to the manufacturers' instructions. The levels of cytokines such as TNF-α, IL-1, IL-6, IL-17, RANKL, MMP-9, and MMP-13 in the culture supernatants from human mast cells were measured using sandwich ELISA (R&D Systems), according to the manufacturer's instructions. Absorbance at 405 nm was also measured using an ELISA microplate reader (Molecular Devices, CA).

The human mast cell line (LUVA) was purchased from Kerafast (Kerafast, MA, USA) and maintained in StemPro-34 SFM at a concentration of 5×10 5 cells/ml. The cells were grown at 37 °C with 5% CO 2 , and the medium was exchanged weekly with 50% fresh medium without exogenous cytokines. The mast cells were seeded in 6-well plates at a density of 5×10 4 cells/ml. After one day, the medium was replaced with StemPro-34 SFM culture medium with or without cytokines. The mast cells were then stimulated with 100 ng/mL human recombinant IL-33 (R&D Systems, Inc.) for 24 and 72 h. The supernatants and RNA were harvested and stored at -80 °C until analysis.
Enzyme-linked immunosorbent assay (ELISA) SF and serum samples obtained from healthy volunteers and patients with RA and osteoarthritis were subjected to tryptase, chymase, and histamine analyses using tryptase (Boster Biological Technology, CA), chymase (Aviva Systems Biology, CA), and histamine (Enzo Life Sciences Inc., NY) ELISA kits, according to the manufacturers' instructions. The levels of cytokines such as TNF-a, IL-1, IL-6, IL-17, RANKL, MMP-9, and MMP-13 in the culture supernatants from human mast cells were measured using sandwich ELISA (R&D Systems), according to the manufacturer's instructions. Absorbance at 405 nm was also measured using an ELISA microplate reader (Molecular Devices, CA).