Nbd pc

We used ϕ6 bacteriophage propagated in the host Pseudomonas syringae as a surrogate for influenza virus. ϕ6 is enveloped and, at approximately 75 nm in diameter [33 (link)], is similar in size. To visualize the virus, we used 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC, Avanti Polar Lipids Inc.), which is assumed to associate with the virus' lipid membrane because of its similar composition.

Lipids dissolved in chloroform were mixed at the desired molar ratio, and the solvent was evaporated by nitrogen gas. The lipid was hydrated with buffer B (20 mM HEPES pH 7.0 and 150 mM NaCl). After five freeze and thaw cycles with liquid nitrogen, liposomes were extruded through a mini-extruder (Avanti Polar Lipids) with 100 nm polycarbonate filter^{56 (link)}.
All lipids used in this study were purchased from Avanti Polar Lipids: PS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), PC (1,2-dioleoylsn-glycero-3-phosphocholine), PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), PA (1,2-dioleoyl-sn-glycero-3-phosphate), PI3P (1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-3′-phosphate)), PI4P ((1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′-phosphate)), Cholesterol, NBD-PE, NBD-PS (1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphoserine), NBD-PC, NBD-PA (1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphate), NBD-Ceramide (N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-D-erythro-sphingosine), NBD-Cholesterol (5-cholesten-3ß-ol 6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproate), Rhod-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)), and DGS-NTA(Ni) (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]).

GUVs were prepared by electroformation using a Nanion Vesicle Prep Pro setup. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid (POPC; Sigma-Aldrich) and 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC; Avanti Polar Lipids) were dissolved in chloroform and mixed in a w/w ratio of 200:1 (POPC:NBD-PC). 100 μl of the lipid mixture at 5 mg ml⁻¹ was spin-coated on the conducting surface of an indium tin oxide (ITO)-coated glass slide (Nanion/VisionTek). Chloroform was evaporated for 1 h in a desiccator following which 600 μl of sucrose buffer (100 mM sucrose, 20 mM HEPES at pH 7.4) was deposited within the O-ring chamber which was subsequently sealed with another ITO-coated slide (conducting surface facing the other). The electroformation chamber was then connected to the Nanion Vesicle Prep Pro and the electroformation protocol proceeded in three steps: (i) The A/C voltage increased linearly from 0 to 3.2 V peak-to-peak (p-p) at 10 Hz over 1 h, (ii) the voltage stayed at 3.2 V p-p and 10 Hz for 50 min, (iii) the frequency decreased linearly to 4 Hz over 10 min and was maintained for another 20 min. Electroformation was carried out at 37 °C and vesicles were stored at 4 °C protected from light. Vesicles were not used longer than 36 h after formation.

Dioleoyl phosphatidylcholine, dioleoyl phosphatidylserine, egg SM, dipalmitoyl phosphatidylserine, GM1, NBD‐PC, and Bodipy‐Chol were purchased from Avanti Polar Lipids. TR–DHPE, AF–CTxB, and OG–DHPE were purchased from Invitrogen. Other chemicals, including NaCl, CaCl₂, MgCl₂, 4‐(2‐Hydroxyethyl)piperazine‐1‐ethanesulfonic acid (HEPES), hexadecane, silicone oil AR 20, methyl‐β‐cyclodextrin, and SMase from Bacillus cereus were obtained from Sigma‐Aldrich. All the lipids were dissolved in chloroform or the mixture of chloroform and methanol, and stored in a refrigerator at −20 °C. NaCl, CaCl₂, MgCl₂, and HEPES were dissolved in distilled water at concentrations of 100 × 10⁻³, 5 × 10⁻³, 2 × 10⁻³, and 10 × 10⁻³m, respectively, to prepare an aqueous buffer solution, followed by titration to pH 7.4 with a 0.5 m NaOH solution. The aqueous buffer solution was stored in the refrigerator at 4 °C. AF–CTxB and SMase were dissolved in the aqueous buffer at concentrations of 50 µg mL⁻¹ and 5 U mL⁻¹, respectively, and they were stored in the refrigerator at 4 °C.

Phospholipase A₂'s (PLA₂) enzymatic activity was evaluated through the hydrolysis of synthetic fluorescent phospholipid, using the fluorescent substrate Acyl 6 : 0 NBD phospholipid, NBD-phosphatidylcholine (NBD-PC) (Avanti Polar Lipids Inc., Alabaster, AL, USA). The assay was performed in a spectrofluorometer (Shimadzu, RF-5301PC, software RFPC) with excitation and emission wavelengths of 460 and 534, respectively. The enzymatic activity of each B. mattogrossensis chromatographic fraction (7, 12, 15, 16, 17, and 20) was evaluated over 250 seconds, after the addition of substrate (3.3 μg/mL, final concentration) in a reaction media containing 50 mM Tris-HCl and 8 mM CaCl₂ at pH 7.5 at room temperature.

The phospholipidosis induced by AM treatment was assessed by the accumulation of a synthetic fluorescent phospholipid analogue, 1-acyl-2-[12-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-glycero-3-phosphocholine (NBD-PC, Avanti Polar Lipids, Inc., Alabaster, AL, USA), according to Kasahara et al. [48 (link)]. In brief, cells were cultured in 96-well black clear bottom plates and treated for 24 h with AM in the presence of 40 μM NBD-PC. After 24 h, cells were washed twice with PBS and NBD-PC fluorescence measured (excitation and emission wavelengths of 485 and 538 nm, respectively) on an Infinite^® 200 PRO microplate reader (TECAN). Cells were then incubated for 20 min at 37 °C with Hoechst 33,342 solution (20 μg/mL in PBS) before measuring Hoechst fluorescence (excitation and emission wavelengths of 355 and 460 nm, respectively). Values for NBD-PC fluorescence were normalized to those of Hoechst fluorescence. Data were reported as folds increased with respect to vehicle-treated cells (0.05% DMSO).