A-23187

The enzyme (native or recombinant) was incubated (FALC Thermoblock) in a buffer solution at 37°C and the reaction was started by addition of 1 mM ATP. For SERCA, the buffer solution contained 80 mM KCl, 25 mM MOPS (pH 7.0 by TRIS), 3 mM MgCl₂, 5 mM sodium azide, 0.2 mM EGTA, 0.2 mM CaCl₂ (about 10 µM free calcium) and 2 µM A23187. In the case of Na,K-ATPase, the buffer solution was composed by 20 mM KCl, 100 mM NaCl, 25 mM MOPS (pH 7.0 by TRIS) and 3 mM MgCl₂. Blank samples were prepared in the absence of calcium ions and in the presence of 2 mM EGTA (SERCA) or in the absence of sodium ions (Na,K-ATPase). Control experiments were performed in the presence of 1 µM thapsigargin (SERCA) or 50 µM ouabain (Na,K-ATPase). The total protein concentration was 10 µg/mL for native and recombinant (WT) SERCA, 1 µg/mL for Na,K-ATPase and 4.5 µg/mL for mutant (D351N) SERCA.
After incubation, aliquots were taken at subsequent times and immediately added to the coloring solution contained in glass disposable test tubes (Corning, mod. 99445-12). Usually, aliquots of 100 µL were added to 900 µL of coloring solution, giving a total final volume of 1 mL. This addition suddenly interrupts the enzymatic ATP hydrolysis, and, therefore, Pi release, due to enzyme denaturation produced by the strong acid conditions.
For each protein, five aliquots, each as triplicate, were taken at different times to plot an activity curve (Pi vs time). Blank was also evaluated as a triplicate. Where indicated, sodium citrate was added from a 10% (w/w) stock 10 minutes after aliquot addition.
The calibration curve was determined by preparing standard solutions of Pi from 1 or 0.1 mM aqueous stock solutions.
All measurements were carried out by an UV/Vis spectrophotometer (Jasco, mod. V-560) provided with a stirring and thermostatable holder (Jasco, mod. EHC-477S). The bandwidth was settled to 2 nm. Acquisition data pitch was 1 s for kinetic measurements and 0.5 nm for spectra registration. Quartz semimicro cuvettes (Hellma, 109.004F-QS) were used to allow continuous stirring of the solution. The temperature was always maintained at 20°C by a Peltier temperature controller (Jasco, mod. EHC-477T).

Non-stimulated bovine PMN (negative control) and PMN confronted with T. gondii tachyzoites (1:2) were fixed at 15, 30 or 60 min of co-incubation depending on the experiment with paraformaldehyde 4% for 15 min at room temperature (RT). As negative control HBSS was used. As a positive control of NET formation, the calcium ionophore A23187 was used at a concentration of 25 µ M. The samples were carefully washed thrice with sterile PBS and incubated in blocking/permeabilization solution (PBS containing 3% BSA, 0.3% Triton X-100; Sigma-Aldrich) for 1 h at RT. Thereafter, the samples were incubated with the primary antibodies indicated in the Table 1 diluted in blocking/permeabilization solution overnight at 4 °C in a humidified chamber. Thereafter, samples were washed thrice in sterile PBS (Sigma-Aldrich) and incubated in secondary antibody solutions (Table 1) for 30 min at RT, protected from light. Nuclear counterstaining was achieved by 4′,6-diamidin-2-phenylindol (DAPI; Sigma-Aldrich) present in mounting medium (Fluoromount G, ThermoFisher).
Images were acquired with a Zeiss Confocal LSM 710 equipped with a motorized XY stage and oil 63× objective (numerical aperture of 1.4) or in a Nikon Eclipse Ti2-A inverted microscope equipped with ReScan confocal microscopic instrumentation (RCM 1.1 Visible, Confocal.nl) and a motorized z-stage (DI1500). Three channels were recorded for signal detection: Blue/DAPI/405-laser, AlexaFluor488/Green/Argon-laser, and AlexaFluor594/Red/HeNe-543 laser. Images were acquired with a digital camera controlled by Zeiss ZEN 2010 software or using the NIS-Elements v 5.11 software (Nikon). Samples were imaged by z-stack optical series with a step-size of 0.3-0.5 microns depth. The z-series were displayed as maximum z-projections, and gamma, brightness, and contrast were adjusted (identically for compared image sets) using Image J software, FIJI version (26 (link)).

Invasion assays were performed as previously described (69 (link)). Briefly, parasites were syringe-lysed through a 27.5-gauge needle, resuspended in Endo buffer and settled onto coverslips with HFF monolayers for 20 min. Endo buffer was then replaced with warm D1 media (DMEM, 20 mM HEPES, 1% fetal bovine serum) and incubated at 37°C for 10 min. Coverslips were fixed and blocked (3% bovine serum albumin [BSA] in PBS), and extracellular parasites were stained with anti-SAG1 antibodies. The samples were then permeabilized (3% BSA, 0.2% Triton X-100 in PBS), and all parasites were stained with the anti-F₁β ATPase antibody and incubated with appropriate secondary antibodies. Parasites were scored as invaded (SAG1–, F₁β ATPase+) or not (SAG1+, F₁β ATPase+) by fluorescence microscopy. These assays were performed in triplicate; at least 10 fields were counted for each replicate with a total of >500 individual parasites per replicate. Significance was determined using multiple two-tailed t tests and the Mann-Whitney test.
For egress assays, parasites were grown on a monolayer on coverslips for 24 to 36 h until most vacuoles contained 16 or 32 parasites. Coverslips were washed twice with prewarmed PBS and incubated with A23187 (or DMSO control) diluted in PBS at 37°C for 2 min. Coverslips were then fixed and stained with rabbit anti-IMC12 antibody. Three replicate coverslips were performed, with at least 10 fields counted for a total of ~200 vacuoles for each parasite strain. Significance was determined using two-way ANOVA of SD-centered means and Tukey’s multiple-comparison test.