Human plasma LDL (Lee Biosciences) was modified using a previously established method.38 (link) The LDL protein concentration was measured using the Lowry Protein Assay (Thermo Fisher Scientific). For eLDL modification, based on the protein concentration, the LDL solution was incubated at 37 °C with different amounts of 0.05% trypsin (Sigma-Aldrich) and cholesterol esterase [Sigma-Aldrich, reconstituted to 1 mg/ml using 0.4 M potassium phosphate monobasic (Sigma-Aldrich)]. Briefly, LDL was first incubated with 7 μg trypsin/mg LDL protein for 6 h, then 12 μg cholesterol esterase/mg LDL protein for 10 h, then trypsin again at 24 μg/mg LDL protein for 6 h, and finally 29 μg cholesterol esterase/mg LDL protein for 48 h. Following the incubations, the LDL solution was dialyzed against PBS for 20–24 h.
For oxLDL modification, 5 mg of LDL was incubated in a 1 ml 10 μM CuSO4 solution at 37 °C for 48 h. Following incubation, the modified LDL solution was dialyzed against PBS for 20–24 h.
TNF-α (Sigma-Aldrich or PeproTech) was reconstituted at 10 μg/ml in sterile PBS, aliquoted, and frozen at −20 °C. Aliquots were used within 6 months of reconstitution.
For oxLDL modification, 5 mg of LDL was incubated in a 1 ml 10 μM CuSO4 solution at 37 °C for 48 h. Following incubation, the modified LDL solution was dialyzed against PBS for 20–24 h.
TNF-α (Sigma-Aldrich or PeproTech) was reconstituted at 10 μg/ml in sterile PBS, aliquoted, and frozen at −20 °C. Aliquots were used within 6 months of reconstitution.
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