Power wave microplate reader

Necrostatin-1 was used to inhibit SeNPs induced necroptosis in PC-3 cells. 3.5 × 10³ cells per well were seeded in Ham’s F-12K (Kaighn’s) medium in 96-well flat bottom cell culture plates. After the resting period of 24 h, cells were subjected with 2 µg Se/ml SeNPs or, 2 µg Se/ml SeNPs and necrostatin-1 (20 μM, or 50 μM) or DMSO, and cultured for 24 h at 37 °C. 10 µl of (5 mg/ml) MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] solution was added to each well, and the plates were incubated for 3.5 h at 37 °C. 80 µl of the solubilizing solution, 20% SDS (w/v) in 50% DMF (v/v), was added to each well in a sterile condition. The plates were kept at 37 °C for 3 h at 120 rpm. 130 µl from each well was transferred into a fresh 96 well plate and analyzed on a BioTek Power Wave Microplate reader at 570 nm. Production of the violet colored formazan in this assay corresponds to the cell viability.

S. aureus HG00123 (link) was grown in TSB or on TSA. Liquid S. aureus HG001 cultures were grown in 96-well plates at 37 °C and under constant agitation using a Biotek powerwave microplate reader.

Clot formation and fibrinolysis were measured in 96‐well plates (Greiner, Stonehouse, UK) with a Powerwave microplate reader (Bio‐Tek, Swindon, UK) as previously described 17. Plasma was diluted to a final concentration of 1 : 6 in TBS. Clot formation for turbidity and fibrinolysis measurements was activated by adding final concentrations of 0.1 U mL⁻¹ human α‐thrombin and 10 mmol L⁻¹ CaCl₂. For fibrinolysis measurements, tissue‐type plasminogen activator was added at a final concentration of 30 ng mL⁻¹. The absorbance (or OD) was measured at a wavelength of 340 nm every 12 s for 3.5 h at 37 °C. Absorbance for turbidity and fibrinolysis measurements was determined in duplicate for all plasma samples. Turbidity profiles were analyzed for maximum absorbance (maximum OD), lag time, time to maximum absorbance, and average rate of clot formation. Fibrinolysis was analyzed for average rate of fibrinolysis and time to half‐maximum absorbance (half lysis).

Serum collected at day 18 of pregnancy was analysed for the presence of IFNγ, IL-1β, IL-6, IL-10, TNF-α together with mouse homologues of IL-8 (KC, LIX, MIP-2) using a multiplex assay according to the manufacturer’s protocol (Milliplex MAP kit, Merck Millipore, Billerica, MA, USA). All standards and samples were assayed in duplicate using a Luminex 200™ System (Luminex Corporation, Austin, TX, USA). Concentrations were determined from standard curves and analysed using xPONENT version 3.1 software (Luminex Corporation).
Mouse C-Reactive Protein was measured in duplicate using a commercially available Enzyme Linked Immunosorbent Assay (R&D Systems, Minneapolis, MN. USA) and optical density (450nm) read on a Powerwave microplate reader (BioTek Instruments, Winooski, VT, USA). Standard curves were generated using KC4 software (BioTek Instruments) and used to determine the concentration of CRP in each sample.