Rhod 5n

Wistar rat pups (newborn P0–1) were obtained from the Valladolid University animal facility. All animals were handled according to the ethical standard of Valladolid University under protocols approved by the animal housing facility and in accordance with the European Convention 123/Council of Europe and Directive 86/609/EEC. Fura-2/AM and Rhod-5N are from Invitrogen (Barcelona, Spain). Fetal bovine serum (FBS) is from Lonza (Barcelona, Spain). Horse serum, neurobasal medium, HBSS medium, B27, L-glutamine and gentamycin are from Gibco (Barcelona, Spain). Papain solution is from Worthington (Lakewood, NJ, USA). The poly-D-lysine and annexin V are from BD (Madrid, Spain). DNase I and antibody against the MCU are from Sigma (Madrid, Spain). IP₃R1 and IP₃R2 primary antibodies are from Santa Cruz Biotechnology (Dallas, TX, USA). IP₃R3 primary antibody is from BD Transduction Laboratories (Madrid, Spain). ER tracker, mitotracker, tetramethylrhodamine, methyl ester (TMRM) and CM-H2DCFDA are from ThermoFisher Scientific. Aβ_1–42 peptide is from Bachem AG (Bubenforf, Switzerland). Other reagents and chemicals were obtained either from Sigma or Merck.

ITC experiments were carried out as previously described (Joung et al., 2012 (link)). Titration of ~50 µM protein (see figure legend) with different ligands were performed at 25°C in 50 mM HEPES (pH 7.4) and 150 mM NaCl, in the presence or absence of 1 mM CaCl₂. Buffer without calcium was prepared as reported (Radhakrishnan et al., 2009 (link)) and checked with rhod-5N (Invitrogen). To obtain the effective heat of binding, results of the titration were corrected using buffer-protein and ligand-buffer controls. Finally, the NITPIC (Keller et al., 2012 (link)) and the Origin9 software (Origin Labs Inc.) were used to analyze these data.
1,2-hexanoyl-sn-glycero-3-phospho-L-serine (C₆-PtdSer) was titrated under critical micellar concentration (CMC) to avoid the formation of C₆-PtdSer micelles (http://www.avantilipids.com) that interfere in the titration.