Lb 941

The cytotoxicity assay used was a fluorescence assay measuring the activity of dead-cell protease released from cells with impaired membrane integrity, which is associated with cell death. The cytotoxicity assay was performed according to the manufacturer’s instructions by using a MultiTox-Fluor Multiplex Cytotoxicity Assay (Promega, USA). Briefly, HUVECs were cultured at a density of 0.5 × 10⁴ cells per well in a 96-well plate in growth medium. Cells were incubated with 7.5% serum from KD patients or patients with bacterial infections in RPMI 1640 medium (Lonza, Tokyo, Japan) for a further 24 h in 96-well plates at 37 °C under a 5% CO₂ atmosphere. HUVECs stimulated with sera from KD patients were treated with IG and/or PSL and incubated at 37 °C under a 5% CO₂ atmosphere for a further 24 h with a final volume of 100 μl per well. Cells were incubated for 1 h with 100 μl per well of 2X MultiTox-Fluor Multiplex Cytotoxicity Assay reagent at 37 °C. The resultant fluorescence from dead cells was measured using a Tristar multimode microplate reader LB 941 (Berthold Technologies) at, 485 nm Ex/520 nm Em. The fluorescence produced was directly proportional to the number of dead cells. Blank values were subtracted, and the increase in activity was calculated on the basis of activity measured from untreated cells. Each sample was measured in duplicate.

Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Dojindo, Kumamoto, Japan). HCAECs were cultured in growth medium in 96-well plates (0.5 × 10⁴ cells/well). Following each experiment, medium was replaced with fresh MV 2 basal medium and the final sample volume adjusted to 100 μL/well. Samples were subjected to MTT assay according to manufacturer instructions and measured in duplicate, using Microplate Reader (Tecan Infinite M200).
Cytotoxicity was evaluated via fluorescence to measure the activity of dead-cell protease, which is released from cells with impaired membrane integrity, using a CytoTox-Glo cytotoxicity assay (Promega, Madison, WI, USA) according to manufacturer instructions. Briefly, HCAECs (0.5 × 10⁴ cells/well) were cultured in growth medium in 96-well plates. After each experiment, medium was replaced with fresh MV 2 basal medium (100 μL/well final volume). Fluorescence measured using a Tristar multimode microplate reader (LB 941; Berthold Technologies, Oak Ridge, TN, USA), was directly proportional to the number of dead cells. Each sample was measured in duplicate.

We established a sandwich ELISA for canstatin (Supplementary Materials [8 (link),27 (link)], Figure S2, Table S1) and the plasma concentration of canstatin was measured. Briefly, the absorbance at 450 and 560 nm of plasma samples diluted at 1:100 in Plasma Sample Diluent (ImmunoChemistry Technologies, Bloomington, MN, USA) was measured by using a TriStar LB941 multimode microplate reader (Berthold, Bad Wildbad, Germany), and the subtracted values [(readings at 450 nm)–(reading at 560 nm)] were calculated. Then, the plasma concentration of canstatin was determined by a regression formula of standard curve created with recombinant mouse canstatin (0.391, 0.781, 1.563, 3.125, 6.25, 12.5 and 25 ng/mL).