Multiskan fc spectrophotometer

The relative concentration of IgG1–IgG4 was determined by ELISA. The assay kit contained two 96-well plates containing immobilized antibodies to IgG1–IgG4. The plate wells were incubated for 30 min at 37 °C with 100 μL IgG diluted with 50-fold serum diluent solution at a 0.1 mg/mL concentration. The plate wells were washed four times with 300 µL of phosphate-salt buffer. An amount of 100 µL of horseradish peroxidase conjugate against human IgG (Vector-Best, Russia, Novosibirsk) was added, incubated for 30 min at 37 °C, and washed four times with 300 µL of phosphate-salt buffer. A 100 µL solution of 3,3′,5,5′-tetramethylbenzidine was added and incubated for 10 min in darkness. The reaction was stopped by adding 100 µL of stop reagent (1.0 M H₂SO₄). Optical density was measured on a Multiskan FC spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) in two-wavelength mode: the main filter of 450 nm and the reference filter of 620 nm. A calibration plot of optical density versus IgG1–IgG4 concentration was plotted in MS Excel. The concentration in the test samples was determine from the calibration graphs. The result was presented as the mean value in a series of three experiments.

A commercial ELISA (Ingenasa^®, INGEZIM FIV, Madrid, Spain) was used and the manufacturer instructions were followed. A ThermoScientific Multiskan FC spectrophotometer was used for optical density readings.

¹H-NMR spectra were acquired on an AVANCE III 400 MHz NMR spectrometer (Bruker, Rheinstetten, Germany) in CDCl₃. Optical rotations were acquired on a Polaar 3005 Polarimeter (Optical Activity, Huntingdon, Great Britain) using a 2.5 cm cell with a Na 589 nm filter and the concentration of samples was denoted as c. Mass spectra data were acquired on a TSQ Quantum Access Max Mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). High-resolution mass spectra (HRMS) were acquired on a LTQ Orbitrap Velos spectrometer (Thermo Scientific) and on a Bruker MicrOTOF. FTIR spectra were acquired on an IR Affinity-1 spectrometer (Shimadzu, Thermo Scientific). Organic solvents used were dried by standard methods when necessary. Commercially available reagents were used without further purification. All reactions were monitored by TLC with silica gel coated plates (EMD/Merck KGaA, Darmstadt, Germany), with visualization by UV light and by charring with 0.1% ninhydrin in EtOH. Column chromatography was performed using Merck 60 Å 70–230 mesh silica gel. The optical density was determined using a Multiskan FC spectrophotometer (Thermo Scientific) at a wavelength of 540 nm when using the MTT assay.

For ALP staining, after induction for 14 days, cells were fixed with 95% ethanol and stained with BCIP/NBT solution according to the manufacturer's protocol (Beyotime Institute of Biotechnology) at room temperature for 2 h. The ALP-positive cells were stained blue/purple. Stained cells were visualized using the Canon IXUS210 camera (Canon, Inc.; magnification). In addition, an Alkaline Phosphatase Staining Kit (BestBio, Inc.) was used to detect the ALP activity, according to the manufacturer's protocol.
For alizarin red staining, after induction for 14 days, cells were washed one or two times with phosphate-buffered saline (PBS), fixed with 95% ethanol for 10 min, washed one or two times with PBS again, covered, and stained with 0.1% alizarin red solution for 10 min. Finally, they were rinsed with PBS and observed under an inverted light microscope. For quantification analysis, 10% hexadecyl pyridinium chloride monohydrate (CPC; Sigma-Aldrich; Merck KGaA) was used to dissolve the mineralized nodules and then the absorbance was measured at 540 nm using a Multiskan™ FC spectrophotometer (Thermo Fisher Scientific, Inc.).

The 86-028NP parent strain or the ΔtoxAvapA mutant were re-suspended from sBHI agar plates grown for 18 hours at 37°C in 5% CO₂ into fresh defined media [16] (link) at an OD₆₀₀ of ∼0.1, then 200 microliters of each re-suspension was placed in duodecuplicate into a sterile non-treated flat-bottomed 96 well plate (#351172, BD Biosciences, Bedford, MA, USA). Empty wells were filled with 200 microliters of sterile water to decrease evaporation, and the plate was covered with sterile gas permeable sealing film (#9123-6100, USA Scientific, Ocala, FL, USA). The plate was incubated for 15 hours with shaking at 35°C in a Multiskan FC spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA), and the OD₅₉₅ was read hourly. Three biological replicates were performed and analyzed by the repeated measures analysis of variance (RMANOVA).

Microtiter plates were coated GII.4-2006b VLPs at 2 μg/ml in carbonate/bicarbonate buffer (pH 9.6) and incubated at 4 °C overnight. Plates were washed three times with PBS-T and blocked for 1 h at 37 °C with PBS-T 3 % BSA. After blocking, plates were incubated with serial dilutions of saliva samples (from 1/20 to 1/160) in PBS-T 1 % BSA for 1.5 h at 37 °C and NoV-specific IgA antibody was detected with a secondary anti-IgA human antibody conjugated with HRP (Sigma) at a dilution of 1/4,000 in PBS-T 1 % BSA for 1 h at 37 °C. As controls, all saliva samples were tested in parallel in wells coated with BSA at 2 μg/ml. To rule out nonspecific binding (false positives), boiled saliva samples were also used as negative controls on wells coated with VLPs. Wells were washed four times with PBS-T, and the bound antibody was detected by the addition of 50 μl of o-phenylenediamine (Sigma). The reaction was stopped at 10 min with 3 M H₂SO₄, and the absorbance was read at 492 nm (Multiskan FC spectrophotometer, Thermo Scientific).

Changes in ATP levels in the PAE-treated A. castellanii were measured using an ATP assay kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. The amoebae were incubated with different concentrations of PAE (50 or 100 μg/mL) and incubated at 25 °C for 24 or 48 h. The cells were rinsed with PBS, resuspended in 100 μL of ATP assay buffer, and homogenized. The supernatant was collected and incubated with ATP reaction mixture at RT for 30 min. Absorbance was measured at 570 nm using a Multiskan FC spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The amoeba cells not treated with PAE were used as the controls. The assays were performed in three independent replications. The results are presented as the mean ± standard deviation (SD) of three individual assays. Statistical analyses were performed using GraphPad Prism 9.1.0 (GraphPad Software, San Diego, CA, USA) and statistical significance was analyzed using one-way of variance (ANOVA) with Dunnett’s post hoc test. The differences in the mean values were considered statistically significant at p < 0.001.