Calcium Ionophores

Fresh buffy coat was obtained from hospital blood bank and subjected for WBC separation using HiSep (Hi-media, Mumbai) and diluted with equal volume of Hank’s balanced salt solution. The metabolism of endogenously bound arachidonic acid to LTB₄ was measured in a total volume of 0.4 ml of the cell suspension at 37°C. The reaction tubes were containing 20 μg arachidonic acid and the investigational drugs (PHF and zileuton). After 45 min pre-incubation the reaction was started by addition of 20 μg of calcium ionophore and 2 μg of glutathione. After 15 min the reaction was stopped with 40 μl of 0.1 M HCl. After centrifugation for 2 min aliquots of the supernatant were subjected to LC-MS/MS estimation of LTB₄.
Hypersil GOLD column (50 mm × 2.1 mm, 1.9 μm, Thermo, Waltham, MA, USA) with a gradient system of acetonitrile with 0.1% formic acid and water with 0.1% formic acid was used for chromatographic separation. The gradient was started with 80:20 water/acetonitrile and reached to 30:70 in 3 min and first line condition was achieved over a period of 2 min. Flow rate was maintained at 0.5 ml/min.
Tandem Mass spectrometric detection of analytes and internal standard (IS) was carried out with an electrospray ionization (ESI) source operated in the negative mode. Multiple Reaction Monitoring (MRM) mode was performed for quantification. 335.2 m/z was selected as precursor ion (Q1) whereas 317.2 m/z and 195.1 m/z (Q3) were selected as product ion for LTB₄. Optimized compound dependent parameters were Declustering Potential (DP) - 76, Entrance Potential (EP) - 10, Collision Energy (CE) - 20 and Collision Cell Exit Potential (CXP) - 8. Probenecid was selected as an IS with 283.91 m/z (Q₁) and 240 m/z (Q3).

A previously developed method for measurement of mitochondrial calcium retention capacity (described in detail in^{19 (link)}) was used to measure the time of yPTP opening after Ca²⁺ addition. Briefly, isolated mitochondria were diluted in CRC buffer (250 mM sucrose, 10 mM Tris-MOPS, 10 µM EGTA-Tris, 5 mM P_i-Tris, 1 µM Calcium Green-5N (Thermo Fisher), 0.5 mg/mL BSA, pH 7.4) to a concentration of 1 mg/mL. The reaction was started by adding 1 mM NADH and 5 µM of Calcium ionophore ETH129. After equilibration for 1 min, 200 µM CaCl₂ was added. The rapid increase in the fluorescence of Calcium Green-5N after sometime was attributed to the release of calcium ions from the mitochondrial matrix into the buffer, likely due to the opening of the permeability transition pore. Matrix swelling was evaluated by measuring optical density changes at 540 nm with a Parkin Elmer Lambda 925 UV–Vis spectrophotometer. Mitochondria (500 µg/mL) were suspended in 2 mL of swelling buffer (150 mM sucrose, 10 mM Tris–HCl, 2 mM KH₂PO₄, pH 7.4) and then, 2 mM CaCl₂ and 10 µM alamethicin were added^{51 (link)}.

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)).

Cells were plated at a density of 300,000 cells/well. To study neuronal activity, neurons were incubated with medium containing pluronic acid (F-127 P3000MP, 1 μm, previously warmed to 36°C) and Oregon Green 488 BAPTA-1 (OGB-488, Fisher Scientific, O6807, 5 μm) on DIV4. Thirty minutes later, the chamber was loaded with crystals coated with the cells, and the dye-containing medium was replaced with fresh medium previously warmed to 36°C. Twenty minutes later, the spontaneous activity of the cells was recorded for 10 min with the N-STORM Super-Resolution System. The following parameters were used: channel intensity of 38% intensity, 30 ms/Hz, 12-bit (no binning), no delay between frames, 20× magnification. Later, the frames were analyzed with a NIS-Element AR microscope, and the fluorescence intensity change (ΔF/F) was calculated for 2 min after acute application of the agonist 2′(3′)-O-(4-benzoyl benzoyl) adenosine 5′-triphosphate triethylammonium salt (BzATP, Sigma, 1 mm). As a control, a calcium ionophore (Sigma, C7522-5MG, 1 mm) was acutely applied, and the fluorescence intensity change (ΔF/F) was calculated for each individual cell.