Ionomycin

Hippocampal neurons were transfected with Ca²⁺ indicators GCaMP6f or R‐GECO1, and mRFP or GFP to identify transfected neurons and presynaptic boutons. Field of views with axonal structures for all conditions were selected based on similar expression levels, while remaining blind for the expression levels of Ca²⁺ indicators. Duo‐color time‐lapses were acquired of 21 frames with 30‐s time interval, with a Z‐stack of three planes with 0.5‐μm interval for each frame, and cells were treated with 1–10 μM ionomycin (Santa Cruz, SC3592) prior to frame 13. Fluorescent intensities of GCaMPf or R‐GECO1 at single boutons were measured for the maximum intensity projections of each frame using a fixed ROI. Fluorescent values were corrected for background fluorescence and normalized to the maximum fluorescent intensity within seven frames after ionomycin treatment (F/F_max). Relative basal Ca²⁺ levels were determined by the F₀/F_max ratio, where baseline values (F₀) were obtained by averaging the fluorescent intensities of the five frames prior to ionomycin treatment.

Blood was collected from 4 female healthy donors (age 26–33 years) as described above, and platelets were isolated as previously described [47 (link)]. In brief, citrated blood was centrifuged at 120xg for 20 min at 20°C to obtain platelet-rich plasma (PRP). Platelets were isolated from PRP by centrifugation at 3,000xg for 2 min at 20°C in the presence of prostacyclin (PGI₂, 1 μM, Sigma-Aldrich, Austria), followed by repeated washing in sterile filtered (0.1 μm) phosphate-buffered saline (PBS: 133 mM NaCl, 1.3 mM KCl, 2.5 mM Na₂HPO₄x2H₂O, 0.5 mM KH₂PO₄, pH = 7.4).
MP generation was induced by addition of thrombin receptor activating peptide-6 (TRAP–6, 20 μM, AnaSpec, Belgium) or ionomycin (40 μM, Santa Cruz Biotechnology, Inc., Germany) in the presence and absence of pPCI (300 nM) to isolated platelets (300,000/μl) in PBS. After 20 min of stimulation platelets were centrifuged at 3,000xg for 5 min to obtain MP containing platelet-free supernatant.

NIH3T3 cells were cultured in DMEM high‐glucose medium with 5% fetal bovine serum, 4 mM l‐glutamine, 200 U/ml penicillin and 200 µg/ml streptomycin at 37°C with 5% CO₂. All imaging experiments with NIH3T3 were done in starving medium consisting of DMEM high glucose supplemented with 0.5% BSA (Sigma), 200 U/ml penicillin, 200 μg/ml streptomycin and 4 mM l‐Glutamine. MCF10A human mammary cells were cultured in DMEM:F12 supplemented with 5% horse serum, 20 ng/ml recombinant human EGF (Peprotech), 10 μg/ml insulin (Sigma), 0.5 μg/ml hydrocortisone (Sigma), 200 U/ml penicillin, and 200 μg/ml streptomycin. All imaging experiments with MCF10A were done in starving medium consisting in DMEM:F12 supplemented with 0.3% BSA, 0.5 μg/ml hydrocortisone, 200 U/ml penicillin, and 200 μg/ml streptomycin. For growth factor stimulations, we used human EGF (AF‐100, Peprotech) and human basic FGF (F0291, Sigma). Chemical perturbations were done with SU‐5402 (SML0443, Sigma), RAF709 (HY‐100510, Lucerna‐Chem), U0126 (S1102, Selleck chemicals, Lubio), SCH772984 (HY‐50846, Lucerna‐Chem), SL0101 (559285, Sigma), Cyclosporine A (10‐1119, Lucerna‐Chem), and Ionomycin (sc‐3592, Santa Cruz). Selection of the cells post transfection was done using Puromycin (P7255, Sigma), Blasticidin S HCI (5502, Tocris), and Hygromycin B (sc‐29067, Lab Force).

To determine intracellular Ca²⁺-concentrations, DAOY cells were seeded on cover slips, mounted in a cell chamber and perfused as described in the section “Whole-cell patch clamp”. Fluorescence was measured every 2 s on an inverted microscope (IX71, Olympus, Chromaphor) using a Fluar 20 × /0.75 objective (Olympus) and Till Vision real-time imaging software (Till Photonics). Cells were loaded for 30 min at 37 °C with 2 μM Fura-2-AM (Molecular Probes) in the bath solution. Fura-2 was excited at 340/380 nm and the emission was recorded between 470 and 550 nm using a sensicam CCD camera (PCO imaging). Acquisition and data analysis were done using the Till Vision software and Excel.
The bath solutions used for the capsaicin, menthol, ATP and ionomycin stimulations consisted of the conditioning bath solution described under “Whole-cell patch clamp” containing the following chemicals: 100 μM capsaicin (Sigma Aldrich), 200 μM menthol (Sigma Aldrich), 100 μM ATP (Sigma Aldrich), or 1 μM ionomycin (Santa Cruz Biotechnology, Dallas, USA), respectively. The 30 mM K⁺ solution contained the following compounds (in mM): NaCl 85, KCl 30, D-glucose 5, HEPES 5, glucose 5.5, MgCl₂ 1, sodium gluconate 25, calcium gluconate 3. pH was adjusted to 7.4 with NAOH and HCl. The pH 6.0 solution consisted of the acidic bath solution described in “Whole-cell patch clamp”.

Reporter assays were performed as previously described60 (link). The Foxp3 promoter (−422- +20, position at Exon1) was cloned into the pGL4.10 Luciferase reporter plasmid (Promega, catalogue number: E6651). The TK-Renilla reporter plasmid (Promega, catalogue number: E2241) was used as a control. Treg cells, polarized for 42 h as described above and expanded for 1 day were transfected with different Luciferase reporter and control Renilla reporter constructs by using the Mouse T Cell Nucleofector Kit (LONZA, catalogue number: V4XP-3032) according to the manufacturer's instructions. Sixteen hours after electroporation, T cells were re-stimulated for 6 h with PMA (25 ng ml⁻¹, Santa Cruz) and Ionomycin (1 μg ml⁻¹, Santa Cruz) and then harvested and measured with the Dual Luciferase reporter system (Promega, catalogue number: E1910). Renilla activity was used to normalize transfection efficiency and Luciferase activity.