Ldh assay

Cytotoxic responses were evaluated according to the manufacturer’s instructions (Promega, Madison, WI, USA). Briefly, splenic CD8⁺ T cells were isolated from mice immunized with PBS or with 10 μg LEX-CD80, LEX-CD86, LEX-CD8086 or LEXnull. Seven days after the last stimulation, splenic CD8⁺ T cells were isolated from immunized mice using an EasySep™ mouse CD8⁺ T cell Isolation Kit (Stem-cell Technologies, Vancouver, Canada). Cells were re-stimulated with irradiated L1210 cells for 7 days and then harvested as effector cells. L1210 cells were used as specific target cells and p388 cells were used as controls. The cells were seeded in a 96-well plate at 1 × 10⁴ cells/well. After adding CytoTox 96^® Reagent (Promega, Madison, WI, USA), absorbance at 490 nm was recorded and cells were incubated for 30 min, protected from light. The magnitude of the cytotoxic response at different effector/target (E/T) ratios was evaluated using a LDH assay (Promega, Madison, WI, USA), and specific lysis (%) was calculated as follows: (experimental LDH release − effector cells − target spontaneous LDH release)/(target maximum LDH release) × 100 [28 (link)].

All reagents unless otherwise specified were from Sigma-Aldrich. Wortmannin, Ly294002, rottlerin, bisindolylmaleimide i, bisindolylmaleimide ix, Gö6983 were from Cayman Chemical. P11, GRGDSP, GRADSP, αvβ3 function blocking antibody (LM609) were from EMD Millipore. PP1, PP2, PP3 were from Calbiochem. LDH assay was from Promega. Phospho-MARCKS Ser152/156 (Cat#2741), MARCKS (Cat#5607), Phospho-PI3K (Cat#4228), p85 PI3K (Cat#4257), Phospho-PKCδ (Cat#2055), PKCd (Cat#9616) antibodies were from Cell Signaling Technology. FITC-PIP3 antibody (Cat#G345) was from Echelon. All secondary antibodies and isotype controls were from Life Technologies.

Cell viability was measured by lactate dehydrogenase (LDH) assay (Promega, Poland). hFOB 1.19 cells were seeded in triplicate at a density of 10⁴ cells/100 μL of cell-culture medium into 96-well plate. The next day, hFOB 1.19 cells were exposure to AgNPs under SF conditions at the concentrations 1, 5, 15, 30, and 60 μg/mL for 18, 24, 48 hours as well as to Ag released from 30 and 60 AgNPs μg/mL (prepared as indicated in section Characterization of AgNPs) for 24 and 48 hours. LDH release into the surrounding medium was measured according to the manufacturer’s protocol. Absorbance values were corrected with blank NPs. LDH data were expressed as a percentage of the total LDH released from cells into the culture medium.