LDL was isolated from blood plasma of healthy donors as described by Van Tits et al. (16 (link)). Briefly, the LDL-Fraction was separated by two 24 h steps of ultracentrifugation at 4°C. After this, 4 dialysis steps against PBS (pH 7.4, 4°C) were performed in increasing time intervals [(1) 1 h, (2) 2 h, (3) 3 h, (4) overnight]. The obtained nLDL was sterile filtered and oxidized with 20 μM CuSO4 for 24 h at 37°C. After oxidation another 4 cycles of dialysis against PBS followed, as described above. OxLDL was sterile filtered and protein concentration was measured with a Lowry Protein Assay (ThermoFisher, 23240) following the manufacturer's instructions. TBARS were measured with OxiSelect TBARS Assay Kit (Cell Biolabs, 1024311). Experiments in the manuscript were performed with low TBAR LDL. To detect possible endotoxin contamination an endotoxin assay was performed with a commercially obtained kit (GenScript, ABIN491527). All nLDL or oxLDL which was used in the experiments had endotoxin levels lower than 0.01 EU/ml.
Lowry protein assay
Manufactured by Thermo Fisher Scientific
The Lowry Protein Assay is a colorimetric method used to quantify the total protein concentration in a sample. It is a commonly used technique in biochemistry and molecular biology laboratories.
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4 protocols using lowry protein assay
1
Isolation and Oxidation of LDL
2
Tumor Cytokine Profile Analysis
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Mice bearing 4T1 breast carcinoma were treated with various therapies stated above, and on day 4 after the completion of treatment, cytokine protein concentrations from tumor nodules were determined by ELISA as described previously.(14 (link)) Tumor tissues were harvested, cut into small pieces, homogenized, and centrifuged at 12 000g for 30 min. Cytokines (granulocyte–macrophage colony-stimulating factor [GM-CSF], γ-interferon [IFN-γ], monokine induced by interferon-gamma [MIG], interferon-gamma-induced protein 10 [IP-10], interleukin-12 [IL-12], transforming growth factor-β [TGF-β], IL-10, and vascular endothelial growth factor [VEGF]) were determined by ELISA in homogenized tumors directly. Tumor-derived cytokine concentrations were corrected for total protein by Lowry protein assay (Thermo), and the results were expressed as pg/mg total protein.
3
LDL Modification and Oxidation Protocols
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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.
4
LDL Modification and Oxidation Protocols
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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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