Atto 594

Treated cells were fixed with 4% paraformaldehyde (AppliChem, Darmstadt, Germany) in PBS for 20 min at room temperature. Fixed cells were washed with PBS, permeabilized with 0.1% Trition X-100 (Bio-Rad Laboratories, Hercules, CA, USA) in PBS for 1 min at room temperature and again transferred to PBS. The actin cytoskeleton was stained using fluorescently labelled phalloidin ATTO 594 (1:200 ATTO-Tec, Siegen, Germany) in PBS for 1 h. The cover slips were mounted in Prolong Diamond antifade mountant with DAPI (inVitrogen, Carlsbad, CA, USA) to stain the nucleus. Pictures were taken with a Axio Vert 135 TV inverse microscope with phase contrast and CoolSnap 4k camera (Zeiss, Oberkochen, Germany). Pictures were processed using Image J (NIH, Bethesda, MD, USA).

Brain SM and DOPC were purchased from Avanti Polar Lipids (Alabaster, AL, USA), and chol was purchased from Sigma Aldrich (St. Louis, MO, USA). Stearoyl-SM (SSM) was purified from Brain SM using a SPD-M10A HPLC (Shimadzu, Shizuoka, Japan) with a 5C18-MS-II C18 reverse-phase column (Nacalai Tesque, Kyoto, Japan) as previously reported^{48 (link)}, with slight modification. Purity was determined by thin layer chromatography with Silica gel 60 F₂₅₄ HX44026754 (Merck, Darmstadt, Germany). 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (Bodipy-PC) was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). 488neg-SM and 594neg-SM, labeled with ATTO488 and ATTO594 (ATTO-TEC GmbH, Siegen, Germany), were synthesized as previously described^{12 (link)}.

cd₁NiR and cNOR were fluorescently labelled using amino-reactive dyes. The protein concentration was set to 3 mg/ml and a 1/20 volume of NaHCO₃ (pH 9.0) was added. cd₁NiR was labelled with a 5-fold molar excess of Abberior STAR 635 (Abberior GmbH) and cNOR was labelled with a 3-fold molar excess of ATTO 594 (ATTO Tec GmbH) by incubating at room temperature while gently shaking for 1.5 h. Unbound dye was removed using a PD-10 column (GE Healthcare), equilibrated with a 10 mM phosphate buffer (pH 7.4) supplemented with 100 mM sucrose, 2 mM KCl and 1 mM (~0.05%) DDM.