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3 protocols using 1 2 dilauroyl sn glycero 3 phosphocholine dlpc

1

DLPC Lipid Preparation and Nile Red Staining

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1,2-Dilauroyl-sn-glycero-3-phosphocholine
(DLPC) that was used for this study was purchased Avanti Polar Lipids.
The phospholipid was dissolved in chloroform (purity >99%). 9-Diethylamino-5H-benzo[α]phenoxazine-5-one
(Nile Red), which was applied as a fluorescence dye, was purchased
from Biotium. All of these chemicals were used without further purification.
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2

Purification and Labeling of Phospholipids

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All chemicals were of analytical grade and purchased from Sigma-Aldrich (Denmark) unless otherwise stated. 2′5′-ADP Sepharose for affinity purification, size exclusion chromatography (SEC) column Superdex 200 HR 10/30 for high-resolution preparative separation, Cy3 and Cy5 maleimide fluorophores and PD-10 desalting columns were purchased from GE Healthcare Life Sciences, Brøndby, Denmark. Phospholipids: 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-dilauroyl-sn-glycero-3-phosphorylglycerol sodium salt (DLPG) were purchased from Avanti Polar Lipids (Alabaster, USA). CriterionTGX Stain-Free (12%) precast gels and Bio-Beads SM-2 were obtained from Bio-Rad.
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3

Cell Culture and Lipid Nanoparticle Preparation

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Human embryonic kidney (HEK) 293T cells, mouse melanoma B16-F10 cells, and mouse embryonic fibroblast BALB/3T3 cells were obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu City, Taiwan, ROC). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Gaithersburg, MD, USA) supplemented with inactivated 10% foetal bovine serum (FBS; Invitrogen) and 1% penicillin–streptomycin amphotericin B (PSA; Biological industries, New York, NY, USA). Human natural killer NK-92 MI cells were grown in alpha minimum essential medium (αMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 0.2 mM inositol (Sigma-Aldrich), 0.2 mM 2-mercaptoethanol (Sigma-Aldrich), 0.02 mM folic acid (Sigma-Aldrich), 12.5% horse serum (Gibco BRL, Gaithersburg, MD, USA) and 12.5% FBS. All cells were incubated at 37 °C in an atmosphere of 5% CO2.
Male C57BL/6 mice (6–8 weeks old) were purchased from the National Laboratory Animal Center (NLAC, Taipei City, Taiwan, ROC).
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids, Inc., Alabaster, AL, USA), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) (Avanti Polar Lipids, Inc.) and polyethylene glycol (PEG, MW 1.500) (Shimo-Meguro, Meguro-Ku, Tokyo, Japan), PEG (MW 8.000, Sigma-Aldrich) and polyethylenimine (PEI, branched, MW 25.000, Sigma-Aldrich) were purchased to prepare LPPC.
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