Kaleido software

The effect of the HA nonwoven textiles on immune cell activation was assessed using the Monocyte Activation Test (MAT). The textile samples were dissolved in normal saline (0.9% NaCl) at a concentration of 1 mg/mL. Heparinised whole blood from four healthy donors was pooled and 10× diluted with normal saline. The diluted blood was placed in sterile microtubes and 100 µL of the sample solution was added, followed by incubation at 37 °C for 16 to 20 h. In parallel, the inner control sample was prepared by further adding Reference Standard Endotoxin (RSE, Merck, Darmstadt, Germany, cat. no. #E0150000) at a concentration of 0.25 EU/mL to the sample solution. The purpose of the inner control sample was to exclude undesired interaction between the sample and the RSE lipopolysaccharide. For evaluation purposes, also solutions of pure RSE at concentrations of 10, 5, 1, 0.25 and 0.1 EU/mL were tested.
After incubation, the microtubes were manually shaken and centrifuged at 13 000 rpm for 10 min. The upper phase (plasma diluted in the saline) was collected for IL-6 analysis using an IL-6 Human Uncoated ELISA kit (Invitrogen, Waltham, MA, USA, #88-7066-88) according to the manufacturer’s protocol. Absorbance was read by an EnSight Multimode Reader and the Kaleido software (both from PerkinElmer, Waltham, MA, USA) was used for evaluation.

The detection of binding events at the equilibrium was performed using the label free, DMR module of an EnSight Multimode Plate Reader (Perkin Elmer, MA, USA) [25 (link)]. Mouse or human recombinant PrP were immobilized onto the surface of DMR plates (15 μL/well of a 2.5 μM recombinant PrP solution in 10 mM sodium acetate buffer, pH 5) using an amine-coupling chemistry. The interaction between CPZ and recombinant PrP or BSA was evaluated by incubating different concentrations of the compound (0.1–2,000 μM; diluted in assay buffer: 10 mM PO4, pH 7.5, 2.4 mM KCl, 138 mM NaCl, 0.05% Tween-20) for 30 min at room temperature. All steps were performed by using a Zephyr Compact Liquid Handling Workstation (Perkin Elmer). Final signals were obtained by automatic intra-well, empty surface normalization, and by subtraction of the control wells (no protein immobilized, or vehicle added). The Kaleido software (Perkin Elmer) was used to acquire and process the data.

The EnSight Multimode Plate Reader (Perkin Elmer) was used to carry out DMR analyses. Immobilization of full-length (residues 23–230) or mouse N-terminally truncated (111–230) recombinant PrP (15 μL/well of a 2.5 μM PrP solution in 10 mM sodium acetate buffer, pH 5) on label-free microplates (EnSpire-LFB high sensitivity microplates, Perkin Elmer) was obtained by amine-coupling chemistry. The interaction between Fe³⁺-TMPyP, SM875 and SM940 diluted to different concentrations (0.03–100 μM, eight 1:3 serial dilutions) in assay buffer (10 mM NaHPO₃, pH 7.5, 2.4 mM KCl, 138 mM NaCl, 0.05% Tween-20) and PrP, was monitored after a 30 min incubation at RT. All the steps were executed by employing a Zephyr Compact Liquid Handling Workstation (Perkin Elmer). Data were obtained by normalizing each signal on the intra-well empty surface, and then by subtraction of the control wells. The Kaleido software (Perkin Elmer) was employed to acquire and process the data.