Nanoparticle Localization in Breast Cancer Cells

Wild type MDA-MB-231 cells or MDA-MB-231 cells stably expressing Rab5-Clover, Rab8a-EGFP, Rab11-GFP, or LAMP1-mGFP were seeded in 8-well Lab-Tek™ chambered coverglasses (VWR, Radnor, PA) to 60%–70% confluence. Cells were incubated with Cy5-labeled FZD7-NS or IgG-NS at a concentration of 2.7 × 10¹⁰ NS/mL for 24 h. GFP-expressing cells were counterstained with 1 μM Hoechst 33342 for 15 min to label cell nuclei. For mitochondria staining, wild type cells were treated with 1 μM Hoechst 33342 and 1 μM Rhodamine123 after the nanoparticles had been applied. Cells were then washed with 1× PBS and imaged with a Zeiss Axioobserver Z1 Inverted Fluorescent Microscope equipped with an apotome using the GFP (intracellular compartment) (excitation, 495 nm; emission, 525/50 nm), Cy5 (NS) (excitation, 615 nm; emission, 675/100 nm), and DAPI (nuclei) (excitation, 395 nm; emission, 445/50 nm) fluorescence channels. For colocalization analysis of antibody-conjugated NS and different subcellular compartments, Manders’ colocalization coefficients [21 ] were calculated for each compartment visualized under the EGFP fluorescence channel using the ImageJ plugin EzColocalization [22 (link)].