Phosphatidylcholine

HPLC grade acetic and formic acid, myricetin-3-O-rhamnoside, quercetin-3-O-rhamnoside, hirsutenone, oregonin, caffeine and rutin standards, dimethyl sulfoxide-d₆ (99.8 atom% D with 0.03 vol.% TMS) and methanol-d₄ (99.8 atom% D), phosphatidylcholine, cholesterol and the porcine polar brain lipid extract were purchased from Merck (Darmstadt, Germany). Ethyl acetate, n-hexane and methanol of reagent grade, HPLC-MS grade acetonitrile, methanol, n-dodecane, dimethyl sulfoxide, NaCl, HCl, Na₂HPO₄·7H₂O and NaH₂PO₄·H₂O were obtained from Reanal-Ker (Budapest, Hungary). HPLC-grade water was prepared with a Millipore Direct Q5 water purification system (Bedford, MA, USA). All aqueous eluents for HPLC were filtered through MF-Millipore membrane filters (0.45 μm, mixed cellulose esters) (Billerica, MA, USA) and degassed in an ultrasonic bath before use.

C-terminally (His)₆-tagged recombinant human Bak(23–185;C166S) and synthetic human Pxt1(76–101; wild-type or DLA) or Bim(141–166) peptides were used in the liposome release assay. To mimic the outer mitochondrial membrane, liposomes were prepared by mixing 46% phosphatidylcholine (Merck), 25% phosphatidylethanolamine (Merck), 11% phosphatidylinositol (Cytiva, US), 10% phosphatidylserine (Alfa Chemistry, US), and 8% cardiolipin (Merck), which were supplemented with 5% nickel-chelating 1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl)iminodiacetic-acid)-succinyl] (Avanti, US) for recruitment of (His)₆-tagged proteins to the membrane. Lipid mixtures in chloroform with 0.01% butylated hydroxytoluene (Merck) were dried under N₂ and resuspended in a buffer containing 10 mM HEPES (pH 7.5), 135 mM KCl, and 50 mM 5(6)-carboxy-fluorescein (Cytiva). Liposomes were then passed over a PD-10 Sephadex G-25 column (Cytiva) to remove the excess dye. Liposomes were incubated with recombinant Bak and/or synthetic peptide in buffer containing 10 mM HEPES (pH 7.5) and 135 mM KCl for 0–90 min at room temperature. Fluorescence was measured by excitation at 485 nm and emission at 535 nm using an Infinite M Plex multimode microplate reader (TECAN, Switzerland).

Analytical grade organic solvents used in this study along with HPTLC chromatography plates (20 × 10 and 10 × 10 cm silica gel 60 F₂₅₄ glass plates) were obtained from Merck (Darmstadt, Germany). Copper sulfate heptahydrate, potassium chloride, orthophosphoric acid (85%, w/w), and sulfuric acid (96% w/w) were obtained from CarlRoth (Karlsruhe, Germany). Lipid standards, namely, phosphatidylcholine (PC 15:0–18:1), phosphatidylethanolamine (PE 15:0–18:1), phosphatidylinositol (PI 18:1–18:1), phosphatidylserine (PS 18:1–18:1), phosphatidylglycerol (PG 16:0–18:1), diacylglyceryl trimethyl-homoserin (DGTS 16:0–16:0), monogalactosyldiacylglycerol (MGDG 18:1–18:1), digalactosyldiacylglycerol (DGDG 18:3–18:3 as main species), sulfoquinovosyldiacylglycerol (SQDG 18:3–16:0 as main species), eicosapentaenoic acid as a free fatty acid (FA), triolein as a triacylglycerol (TAG), di-oleic acid as a diacylglycerol (DAG), and monooleic acid as a monoacylglycerol (MAG), were purchased from Merck. The lipid standards were dissolved in n-hexane (p.a. ≥ 99%) and sonicated for 30 s. Stock solutions were prepared at a concentration of 1 mg mL⁻¹ and aliquoted into 1.5-mL vials under a nitrogen stream, sealed, and stored at − 85 °C until analysis.

Tris(hydroxymethyl)aminomethane (Tris), Guanidine-HCl, NaCl, KCl, CaCl₂, MgCl₂, DTT, EGTA, β-mercaptoethanol, NH₄HCO₃, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), Ames’ medium, ethanolamine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, cholesterol, acrylamide, Coomassie blue, cGMP, polyethyleneimine, sucrose, OCT, NH₄Cl, citric acid, Triton X-100, Tween 20, Bovine Serum Albumin, chloramphenicol, cOmplete EDTA-free Protease Inhibitor Cocktail, paraformaldehyde, ketamine, xylazine, atropine, hydrocortisone and BaCl₂ were purchased from Merck (Darmstadt, Germany).
DMEM, OptiMEM, penicillin, streptomycin, 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI), Phosphate Saline Buffer (PBS), Fetal Bovine Serum (FBS), HBSS, glutamine, Normal Goat Serum, Normal Donkey Serum were purchased from ThermoFisher Scientific (Waltham, MA, USA).

The Lyosecretome production was made at an accredited facility to produce and control veterinary drugs for clinical use (Istituto Zooprofilattico Sperimentale Lombardia and Emilia-Romagna (IZSLER, Brescia, Italy). The production protocol has been approved by the Italian Ministry of Health (Prot. n. 0000778 del 15/01/2020 7.1.2.0.0.0/17/2019—AGD 809). Culture media, trypsin, and antibiotics used for cell cultures were purchased from Euroclone (Milan, Italy). A commercial platelet lysate kit (PL) was obtained from Carlo Erba Reagents (Milan, Italy). Fetal Bovine Serum (FBS) was purchased by Gibco-Thermofisher (Milan, Italy). Chemicals such as mannitol, bovine serum albumin, Nile Red, acetone, collagenase, and phosphatidylcholine were obtained from Merck Life Science S.r.l. (Milan, Italy) unless mentioned.