Eight chamber

CHO cells were maintained in growth media (F-12K + 10% FBS and Penicillin/ Streptomycin) on glass chamber slides for 24 h (eight chamber, Nunc). The cells were then washed with DPBS once and incubated with Ag-488 or QD-488, with or without T-Ag in DMEM medium for 1 h at 37 °C. Here, 2x concentration of NPs was used compare to flow cytometry study. After incubation, cells were etched for 1 min, aspirated, then washed with DPBS twice and fixed with 4% PFA. The chamber slides were then mounted in DAPI-containing mounting medium (Vector Laboratories, Burlingame, CA) with a coverslip and examined under a Zeiss LSM 710 NLO confocal microscope. Cells were also incubated in AA-free and Cys medium mixed with Ag-488 and T-Ag and treated similarly to image.

Cells were grown on glass chamber slides containing 0.05 × 10⁶ cells per chamber (eight chamber, Nunc, Scotts Valley, CA, USA) for 24 h. Cells were then washed by DPBS buffer once and incubated with AgNP-555 or AgNP-555 + TP peptide in serum-free DMEM medium at 37 °C. 1 h later, cells were etched and washed with PBS. To stain early/late endosome markers, cells were first treated with PBS containing 1% BSA and 0.1% Trition X100 (blocking buffer) at room temperature for 1 h. Cells were washed with PBS and then incubated with primary antibodies diluted (1:200) in blocking buffer at 4 °C overnight, followed by appropriate secondary antibodies diluted (1:200) in blocking buffer at room temperature for 1 h. After PBS washing, cells were fixed with 4% PFA. The chamber slides were mounted in DAPI-containing mounting medium (Vector Laboratories, Burlingame, CA, USA) by coverslips (Leica, Buffalo Grove, IL, USA, Cat# 3800145ACS). Nikon C2 confocal (Melville, NY, USA) was used here for imaging.