Cell lytic buffer

To quantify uptake, cells were exposed to increasing concentrations of Qdots for 24 h. Cells were then washed and lysed in cell lytic buffer (Sigma, St Louis, MO) for 10 min at 4°C. Lysates were centrifuged at 50,000 x g for 1 h at 4°C to pellet internalized Qdots. Pellets were resuspended in water and assayed for cadmium levels using inductively coupled plasma-mass spectroscopy (ICP-MS; Agilent 7500ce, Agilent, Inc., Santa Clara, CA) following EPA Method 6020A.
To visualize Qdot uptake, cells were seeded onto glass chamber slides, allowed to adhere for 24 h, exposed to Qdots for 24 h, fixed with cold methanol for 10 min, and washed 3 times with PBS. Slides were mounted with ProLong Gold anti-fade reagent containing 4′, 6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA). Images were collected using a 490ex/525em LP dichroic cube with a Nuance multispectral camera (Caliper Life Sciences, Hopkinton, MA) mounted on an Optiphot microscope (Nikon, Melville, NY). Differential interference contrast (DIC) images were collected simultaneously with fluorescent digital images.

Macrophages were lysed with Cell Lytic Buffer (Sigma) and the cell lysate was collected. For p47 ^phox estimation the cell lysate was centrifuged at 600g for 10 min at 4°C to remove unbroken cells and nuclei. The supernatant was then ultracentrifuged at 100,000g for 1hr at 4°C to isolate the membrane fraction. Protein was estimated by Bradford’s reagent and equal amounts of protein sample from each experimental condition was subjected to immunoblot analysis using the following primary antibodies: goat poly anti-MCPIP (1:500), rabbit anti-IRE-1 (1:500), rabbit anti-LC3 II (1:500), rabbit anti-C/EBPβ (1:100), rabbit anti-PPARγ (1:100), rabbit anti-FIZZ1 (1:500), monoclonal anti–p47^phox (1:200), and rabbit anti-Fas (1:1000; Santa Cruz Biotechnology); mouse polyclonal anti-GAPDH (1:1000); rabbit anti-GRP78 (1:500); rabbit anti-BECLin 1(1:1000), goat anti-Arg1 (1:2000; Cell signaling). The immune complexes were detected autoradiographically using appropriate peroxidase-labeled secondary antibodies (Santa Cruz Biotechnology) and enhanced chemiluminescence detection reagent ECL (GE Healthcare). Anti-β-actin and anti-GAPDH antibodies served as loading controls. Specific bands were quantified by densitometry using analytic software (Image J) and expressed as a ratio over loading controls.