Slt rainbow plate reader

The substrate Z-L-Lys-SBzl hydrochloride (Sigma, St. Louis, MO) was added at different concentrations (19.5-2,500 µM to measure Vmax and Km, and 2,500 µM for GzmA-S195A experiments) and diluted in assay buffer (50 mM Tris, 154 mM NaCl, pH 7.5) in presence of 0.55 M (5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) (Sigma, St. Louis, MO) chromophore. 120 pM of protein was added per well and substrate hydrolysis was quantified by measuring the absorbance at 405 nm using an SLT Rainbow plate reader (Tecan, Männedorf, Switzerland). Esterolytic activity was reported as rate of hydrolysis using extinction coefficient of 13,100 M^-1cm^-1 for the 3-carboxy-4-nitrophenoxide ion. Specific activity measured as nM product/min/nM of enzyme present.

Serum ATX levels were determined with the Quantikine human ATX immunoassay (R&D Systems, Minneapolis, Minnesota), according to the recommendation of the manufacturer. All measurements were performed on a Tecan SLT Rainbow Plate Reader (Tecan, Männedorf, Switzerland).

25(OH)D₃ levels were quantified by radioimmunoassay (I125 Radioimmunoassay IA Kit; DiaSorin, Stillwater, MN) on a Multi Crystal LB2111 Gamma Counter (Berthold, Bad Wildbad, Germany) from serum samples obtained at the day of study inclusion as described recently [19 (link)]. Samples were blinded for the person who performed measurements, and all measurements were determined at the same day from prospectively collected and stored serum samples. Serum 25(OH)D₃ concentrations <10 ng/ml were defined as severe vitamin D deficiency; levels from 10 to 20 ng/ml were considered as insufficiency and serum levels >20 ng/ml were considered normal. Serum soluble CD163 (sCD163) concentrations were assessed with the Macro163 sandwich enzyme-linked immunosorbent assay (ELISA) (Trillium Diagnostics, Bangor, ME) according to the recommendation of the manufacturer as described previously [13 (link)]. Samples were measured in duplicates on a Tecan SLT Rainbow plate reader (Tecan, Männedorf, Switzerland).