Polarstar omega spectrophotometer

Intracellular calcium concentration was measured as previously described^{59 (link)}. Briefly, cells were pre-transduced with adenovirus encoding cytosolic aequorin at 5 MOI for 36 h, then seeded in white 96-well plate at a density of 10,000 cells/well and incubated overnight. Cells were incubated with 100 μl of modified KRB (0.5 ml of 1 M CaCl₂ was added to 500 ml of KRB) that contained 15 μM coelenterazine (Cat. No. 303-500; NanoLight Technologies, Pinetop, AZ, USA) for 1 h at 37 °C in a 5% CO₂ incubator. Luminescence emission representing free intracellular calcium concentrations were obtained by a POLARstar Omega spectrophotometer (BMG Labtech, Cary, NC, USA). Relative intracellular calcium was expressed as percentage of control.

Unless otherwise stated, all enzymes were assayed in a 200 μL reaction volume using the sodium acetate reaction buffer (100 mM sodium acetate, 1 mM EDTA, 1 mM DTT, 0.01% [w/v] brij L23, pH 7.0). Enzymatic concentration and substrates used in the screening assays are presented in Table 3. Initially, the reaction buffer was mixed with the propeptide inhibitor and the peptidase target was then added to the reaction and incubated for 10 min at 37 °C before the fluorogenic substrate was added. The broad-spectrum inhibitor E-64 (100 μM; Sigma-Aldrich) was used as a positive control inhibitor of cysteine peptidases. Hydrolytic activity was measured over 1 h, at 37 °C as relative fluorescent units (RFU) in a PolarStar Omega Spectrophotometer (BMG LabTech). All assays were carried out in triplicate.
Table 3

Assay conditions of each cysteine peptidase screened against the wild-type ppFhCL3, ppFhCL3 variants and synthetic peptide

Enzyme	Substrate
F. hepatica cathepsin L1	Z-Leu-Arg-NHMec (20 μM)
(FhCL1; 2.7 nM)
F. hepatica cathepsin L2	Z-Leu-Arg-NHMec (20 μM)
(FhCL2; 5 nM)
F. hepatica cathepsin L3	Z-Gly-Pro-Arg-NHMec (20 μM)
(FhCL3; 5 nM)
Human cathepsin L	Z-Phe-Arg-NHMec (20 μM)
(HsCL; 0.2 nM)
Human cathepsin K	Z-Phe-Arg-NHMec (20 μM)
(HsCK; 2 nM)

Explant viability of un-inoculated wound tissue explants at days 1, 3 and 5 were assessed using the XTT cell proliferation assay^{27 (link)}. The second generation colourless or slightly yellow tetrazolium dye is reduced to a soluble brightly-coloured orange derivative. This is achieved by breaking apart the positively charged quaternary tetrazole ring by a mix of cellular effectors including mitochondrial activity^{41 (link)}. A saturated solution was prepared from XTT reagent (Sigma-Aldrich, Poole, UK) in 1 × PBS at a concentration of 0.5 g/L. To activate XTT, 100 µl of 1 mM menadione (Sigma-Aldrich, Saint Louis, USA) was added to every 10 ml of XTT solution used. Wound explants were placed into a sterile 24-well Corning® Costar® cell culture plate and 400 µl/well activated XTT solution was added. Following incubation for 2 h at 37 °C in the dark, the colour change was measured using a POLARStar Omega spectrophotometer (BMG Labtech, Ortenberg, Germany) at an absorbance of 490 nm. Results were processed using Omega software (BMG Labtech, Ortenberg, Germany). Triplicate samples for each time point and wound model were prepared and duplicate independent experiments were performed.

Overnight cultures of individual strains were spun down (5,000 × g, 5 min), resuspended to an OD₆₀₀ of 1.0 in PBS-T, and transferred in 500 µl aliquots into 2 ml microcentrifuge tubes. 50 µg of GFP-tagged RBP was added to the cells and incubated for 90 min on an overhead rotator. Cells were spun down and washed once with 1 ml PBS-T and resuspended in 200 µl PBS-T. 150 µl of the cell suspension was pipetted into a well of a black, flat bottom 96-well microplate (Greiner Bio-One, Austria) and the fluorescence intensity of bound GFP-RBP was measured at ambient temperature using a POLARStar Omega spectrophotometer (BMG Labtech, Germany) at 485 nm excitation, 520 nm emission with (1000 x) fixed gain. Fluorescence binding was performed in triplicate with mean (raw fluorescence) ± standard deviation. For fluorescence microscopy, 4 μl of the cell suspension was imaged using a confocal inverted microscope (Leica TCS SPE) equipped with an ACS APO 63×/1.30 oil CS lens objective with excitation at 488 nm and emissions collected with a PMT detector in the detection range of 510 to 550 nm. Transmitted-light microscopy images were obtained with the differential interference contrast mode. Images were acquired with a Leica DFC 365 FX digital camera controlled with the LAS AF software. Fiji v2.0.0 (ImageJ software) was used to generate final images.

The enzymatic activity of the recombinant FhSODs was measured via the adaptation of an existing xanthine oxidase (XOD)-based SOD assay, whereby O₂^•- is enzymatically produced because of the conversion of xanthine to H₂O₂ and uric acid, which in turn transforms nitroblue tetrazolium (NBT) to NBT-diformazan dye [27 (link)]. To determine the activity of our recombinant enzymes under physiological conditions compatible with the parasite, our assay was conducted at 37 °C in 200 µL assay buffer (1 × PBS; Sigma-Aldrich, 0.1 mM Hypoxanthine; Sigma-Aldrich, 0.1 mM DTPA; Sigma-Aldrich, 2.5 µL/mL tetrazolium salt; Cayman Chemical, Ann Arbor, MI, USA). All other enzymes (native XOD from bovine milk; Roche, used at a final concentration of 8.0 × 10⁻³ U/mL), including the standard curve (native bovine erythrocyte SOD, BS; Sigma-Aldrich), were diluted in PBS prior to use. Recombinant proteins and the standard curve (BS) were serially diluted 1:1 in PBS (rFhSOD1 and rFhSOD3: 10–0.3125 μg/mL; BS: 100–3.125 μg/mL, corresponding to 4–0.125 U/mL) and run in duplicate at a final assay volume of 250 μL. The assay was read continuously for 30 min at 450 nm using a microplate reader (PolarStar Omega Spectrophotometer; BMG LabTech, Ortenburg, Germany) immediately after the addition of XOD.