Effects of TMAO on cholesterol loading of macrophages were conducted without radiolabeled cholesterol in parallel with
cholesterol efflux assays. Lipids were extracted from cells, and total cholesterol mass was determined using Cholesterol
E kit (Wako Diagnostics, USA).
The fraction of cholesterol mass present as free cholesterol (FC) and esterified cholesterol (CE) in cells was calculated
by multiplying the total cholesterol mass by the fraction of cellular [3H]-cholesterol present as FC and CE.
Cellular protein was determined using Markwell-modified Lowry assay [37].
All cholesterol mass determinations (mg/mg cell protein) are expressed as average±standard deviation of triplicate measurements.