Culturing of THP-1 monocytes and their differentiation into macrophages was carried out as previously described [6 (link),11 (link),40 (link),41 (link)]. Primary HASMC were cultured in smooth muscle cell growth medium, as described by the manufacturer (Sigma-Aldrich) [6 (link)]. Culturing of HMDM from buffy coats obtained from the National Blood Service, Wales, was performed, as in our previous study [6 (link)]. Each donor provided informed consent for use of human blood for non-transfusion purposes, and all experiments and associated ethical clearances were approved by Cardiff University.
Determination of cell viability by following the release of LDH into the medium, cell proliferation using crystal violet, rate of cell proliferation using the BrdU labelling and detection kit III, MCP-1-driven monocytic migration, uptake of Dil-labelled oxLDL, macropinocytosis using Lucifer yellow, phagocytosis using a Vybrant® Phagocytosis Assay Kit, and radioactive-based cholesterol efflux from foam cells to ApoA1 acceptors were performed, as previously described [6 (link),11 (link),41 (link),42 (link),43 (link),44 (link)]. ROS were measured using a 2′7′-dichlorofluorescin diacetate (DCFDA) Cellular ROS Detection Assay Kit (ab113851), according to the manufacturer’s instructions (Abcam), with TBHP (50 μM) used to produce ROS, as seen in pathological conditions [6 (link),45 (link)]. PDGF-BB (designated as PDGF)-induced migration of HASMC was performed using a modified Boyden chamber with Matrigel-coated membrane, as in our previous studies [6 (link),11 (link)].
Determination of cell viability by following the release of LDH into the medium, cell proliferation using crystal violet, rate of cell proliferation using the BrdU labelling and detection kit III, MCP-1-driven monocytic migration, uptake of Dil-labelled oxLDL, macropinocytosis using Lucifer yellow, phagocytosis using a Vybrant® Phagocytosis Assay Kit, and radioactive-based cholesterol efflux from foam cells to ApoA1 acceptors were performed, as previously described [6 (link),11 (link),41 (link),42 (link),43 (link),44 (link)]. ROS were measured using a 2′7′-dichlorofluorescin diacetate (DCFDA) Cellular ROS Detection Assay Kit (ab113851), according to the manufacturer’s instructions (Abcam), with TBHP (50 μM) used to produce ROS, as seen in pathological conditions [6 (link),45 (link)]. PDGF-BB (designated as PDGF)-induced migration of HASMC was performed using a modified Boyden chamber with Matrigel-coated membrane, as in our previous studies [6 (link),11 (link)].
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