Nanomaterial Uptake and NF-κB Activation

Raw 264.7 cells preseeded onto a 8-well Nunc Lab-Tek Chamber Slide system and incubated overnight until ~70% confluence were incubated with either amino-f-Sc₃N@C₈₀ or amino-f-Gd₃N@C₈₀ at 1 μM overnight and then stimulated with or without LPS (100 ng/mL) for 30 min. Cells were gently washed with warm PBS and fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature, washed with PBS and then permeabilized with 0.3% Triton X-100 in PBS for 10 min, blocked with 3% bovine serum albumin (BSA), and incubated with rabbit p65 (1:500, Cell Signaling Technology) at 4 °C overnight. Then cells were incubated with goat antirabbit AlexaFluo594 (1:1000, Life Technologies, Carlsbad, CA) at room temperature for 1 h and mounted with Prolong Gold antifade reagent (with DAPI). IgG control staining was performed with identical procedures excluding the primary antibodies. Fluorescence images were taken with a Nikon ECLIPSE E600 microscope with Zeiss software (Nikon Instruments Inc., Melville, NY) as we established.^{12 (link)} At a magnification of 200×, all cells were imaged with identical settings. Total cells (DAPI) and p65 nuclear location were counted from at least four images per chamber per condition in triplicated experiments with ImageJ. The number of total cells was enumerated by a customized ImageJ Macro (see Supporting Information).