Figure 3

Mesenchymal stem cell endocytose low density lipoprotein (ox-LDL) through scavenger receptors, and internalized ox-LDL colocalizes with phagosomes of gfpMtb. (a,b) Naïve MSCs were incubated with red fluorescent Dil-labeled oxidized low density lipoprotein (LDL) ligand (Dil-oxLDL) for SRs. At the time points indicated, the cells were chased with warm medium, and qualitative and quantitative fluorescence readouts were determined. (a) Fluorescence deconvolution images examined using Nikon microcopy (NIS-Elements AR deconvolution software) show endosomes containing Dil-oxLDL. (b) MSCs were read for intracellular fluorescence using an Ascent fluorometer (530nm-ex/599nm-em) and average fluorescence units (AFL) were plotted using triplicate wells per group in 3 replicate experiments. The uptake of Dil-oxLDL increased over 24 hr and then declined. (c) MSCs were treated with Dil-oxLDL for 90 min, washed and infected with gfpMtb (MOI = 1), and incubated for 4 or 24 hrs. Cells were washed, fixed and imaged using deconvolution. The percentage of MSCs containing gfpMtb phagosomes colocalizing with Dil-oxLDL (arrows; inset) was determined, and representative images shown are from one of three identical experiments, each of which counted and averaged 150 cells (N = nucleus; bar = 5 µM). (d) Quantification of MSCs showing Dil-oxLDL and gfpMtb colocalization (**p < 0.009, t test).