Evaluating NF-κB Activation by AuNPs

As described by Schwarz et al.,^{30 (link)} HEK293 cells (mycoplasma negative, culture passage 5–15) were seeded in DMEM [DMEM (Sigma-Aldrich) supplemented with 10% FBS, 2 mM MEM non-essential amino acids (PAA) (Sigma-Aldrich), 2 mM l-glutamine, 100 U mL⁻¹ penicillin/streptomycin] at a final concentration of 1.5 × 10⁵ cells per mL and incubated for 24 h. On day 2, cells were transfected with both a reporter plasmid containing the NF-κB transcription factor linked to a luciferase reporter gene, and a mix of 3 plasmids (TLR4, MD2 and CD14) encoding the LPS receptor components. After 24 h, supernatants were discarded and substituted with 900 μL of fresh medium. Cells were then stimulated by the addition of LPS 30 pg mL⁻¹ (E. coli lipopolysaccharide (LPS) 055:B5 (Sigma-Aldrich)), AuNPs of different sizes (5 × 10¹¹ NPs per well), LPS plus AuNPs, or left untreated. Volume differences were compensated by adding PBS to a final volume of 1.2 mL. On day 4, supernatants were discarded, cells were lysed and lysates were read by a Tecan Infinite M200Pro instrument. To exclude AuNP interference in signal absorbance, data are expressed in the form of a ratio between LPS or AuNPs + LPS and the corresponding controls, untreated cells or AuNPs.