Uptake of Dil-Ac-LDL by endothelial cells

• Cells are seeded in a 24-well plate coated with gelatin-based coating solution (catalog # 6950, Cell Biologics) for 5-10 minutes to 70–90% confluence.
• Wash cells two times with 1% BSA in 1X PBS with Calcium & Magnesium (wash buffer).
• Incubate cells with cell culture medium containing 0.05% FBS for 2 hours in an incubator (37⁰C).
• Remove cell culture medium from the plate.
• Incubate endothelial cells with Dil-Ac-LDL at the concentration of 10 ug/ml for 4 h at 37°C. Note, mix Dil-Ac-LDL (Catalog No. L-35353/Alexa 594, Life Technologies) with complete cell culture medium containing 2-10 % FBS.
• Decant the medium and wash the cells 3 times with wash buffer, 3 min each wash.
• Keep the cells with 0.5-1.0 ml medium in the dark to analyze under a fluorescence microscope ASAP between 5 min - 4 hours (or fix cells and keep cells at 4°C in the dark until the scheduled time for analysis before 12-24 hours).
• For the best result, analyze the cells as soon as possible after staining procedure.

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