Atto 565 dppe

To prepare the lipid stock solution, E. coli polar lipid extract (Avanti Polar Lipids, Inc.) was dissolved in water and sonicated for 30 min to make a 30-mg/mL aqueous suspension stock. The lipid stock solution was flash frozen in liquid nitrogen and stored at −80°C. To prepare a 2 mg/mL aqueous suspension stock of lipopolysaccharide, LPS from E. coli EH100 (Ra mutant; Sigma) was dissolved in water and sonicated for 30 min. The lipopolysaccharide stock solution was flash frozen in liquid nitrogen and stored in aliquots at −80°C.
To prepare the fluorescent lipid stock solutions for flow cytometry, E. coli polar lipid extract (Avanti Polar Lipids, Inc.) was dissolved in chloroform with 1% (molar ratio) ATTO-488 DPPE, ATTO-565 DPPE, or ATTO-647N DPPE (ATTO-TEC GmbH). Lipid mixtures were then dried into a film, hydrated, and lyophilized overnight. The lipid mixtures were then dissolved in water and sonicated for 30 min to make a 30-mg/mL aqueous suspension stock. The lipid stock solution was flash frozen in liquid nitrogen, and stored at −80°C.

1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, 16:0(3me,7me,11me,15me)), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, C16), and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). The photoswitchable ceramide PhoDAG-1 [27 (link)] was synthesized as previously described [27 (link)]. Labeled phospholipids Atto565 DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, C16) and Atto633 PPE (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, C16) were obtained from ATTO TEC GmbH (Siegen, Germany) and used for visual differentiation of the two leaflets. The aqueous solution contained 20 mM HEPES and 20 mM KCl (pH = 7.0). Hexane and hexadecane were from Merck (Darmstadt, Germany).