Glomax luminometer system

100 μL of Parental Raji cells or SLC1A1 knockout Raji cells were seeded at 0.5 million cells/mL in a 96 well plate the evening before activation. The next morning, cells were either left unstimulated or 20 μg/mL of F(ab’)2 anti-human IgM (Southern Biotech) was added directly to the well. 48 hours later, intracellular glutathione levels were measured with the GSH-Glo^™ Glutathione Assay kit (Promega), following the manufacturer’s protocol. Luminescence was recorded with a 0.3 second integration time on a GloMax luminometer system (Promega).

HUVEC (EMD Millipore, Billerica, MA, USA) were maintained in an EndoGRO™ -MV-VEGF Complete Media Kit (EMD Millipore). Cells were seeded at a density of 5,000 cells/well into 96-well plates. After 24 hours, cells were treated for a further 24 hours with different concentrations of SiNPs (0.1–100 μg/mL) in triplicate. Control cultures were treated with saline. The effect of SiNPs on cell viability was determined using a CellTiter-Glo^® Luminescent Cell Viability Assay (Promega Corporation, Fitchburg, WI, USA), based on quantification of adenosine triphosphate, which signals the presence of metabolically active cells. The luminescent signal was measured using the GloMax^® Luminometer system (Promega Corporation). Data were presented as proportional viability (%) by comparing the treated group with the untreated cells, the viability of which is assumed to be 100%.

100 μL of Parental Raji cells or SLC1A1 knockout Raji cells were seeded at 0.6 million cells/mL in a 96 well plate 3 hours before activation. Cells were either left unstimulated or 20 μg/mL of F(ab’)2 anti-human IgM (Southern Biotech) was added directly to the well. At 2 minutes, 10 minutes, or 1 hour after activation, cells were harvested at 300 × g for 3 minutes at 4°C and then washed twice with ice-cold PBS. Cells were then resuspended in 25 μL ice-cold PBS, then 12.5 μL of Inactivation Solution (0.6N HCl) was added to each well to rapidly stop metabolism and destroy reduced NAD(P)H dinucleotides. Five minutes later, 12.5 μL of Neutralization Solution (1M Tris Base) was added to each well, and then 50 μL of Glutamate Detection Reagent (Promega, Glutamate-Glo Kit) was added to each well, and the plate was incubated for 60 minutes at room temperature. Luminescence was recorded with a 0.3 second integration time on a GloMax luminometer system (Promega).