96 well plate

In 2D culture, 2800 cells/well were seeded into a 96-well plate (Corning, USA) coated with different hydrogels (Col I, CSM/Col I or BMP2-CSM/Col I hydrogel). In 3D culture, Col I, CSM/Col I and BMP2-CSM/Col I hydrogels with 1 × 10⁶/ml cells were added into a 96-well plate (Corning, USA) respectively, and then placed in a cell incubator. Culture medium was added after gel formation and changed every day. Cell proliferation was evaluated using CCK-8 at Days 1, 4, 7 and 10.

B16 F10 melanoma cell and MDCK-GFP cell suspension (10000 cells/well) was dispensed in a 96-well plate (Corning) and incubated for 12 h at 37 °C under 5% CO₂. Then, cells were co-incubated with 20 nM CytC/ssAuNPs-AuNPs and ssDNA-AuNPs for 12 h at 37 °C under 5% CO₂. After that, the Cell Counting Kit-8 solution (CCK-8, Dojindo Laboratories, Kumamoto, Japan) was added to each well of the plate. After further incubation for 2 h, absorbance at 450 nm was measured using a microplate reader. Results are expressed as the ratio of the absorbance of the positive control, no gold nanoparticle treated cells. To determine the cancer ablation rate with CytC/ssDNA-AuNPs under light irradiation, B16 F10 melanoma cell suspension (10000 cells/well) was dispensed in a 96-well plate (Corning) and incubated for 12 h at 37 °C under 5% CO₂. Then, cells were co-incubated with 20 nM CytC/ssAuNPs-AuNPs for 12 h at 37 °C under 5% CO₂. After that, 660 nm laser with 14 W/cm² was irradiated to each well of the plate for 5 min and the Cell Counting Kit-8 solution (CCK-8, Dojindo Laboratories, Kumamoto, Japan) was added to each well of the plate. After further incubation for 2 h, absorbance at 450 nm was measured using a microplate reader. Results are expressed as the ratio of the absorbance of the positive control, no gold nanoparticle treated cells.

Hydrogen peroxide (Sigma, St. Louis, MO, USA) was serially diluted in Sabouraud dextrose broth (SDB) (Oxoid, Hampshire, UK) on a 96-well plate (Corning, Corning, NY, USA) to produce a concentration range between 30.6 and 245 mM. Amphotericin B (Sigma, St. Louis, MO, USA) and itraconazole (Sigma, St. Louis, MO, USA) were serially diluted in SDB, producing ranges of 0.78 to 62.5 mg/mL and 0.78 to 6.25 µg/mL, respectively. Conidia from the control and passaged strains were harvested and enumerated. Aliquots (100 μL) of conidia were added to each well of a 96-well plate (Corning, Corning, NY, USA) to provide a concentration of 1 × 10⁵ conidia per well. Plates were incubated at 37 °C and growth was assessed at 24 h at 600 nm using a plate reader (Bio-Tek Synergy HT, Somerset, NJ, USA).

Sera were heat-inactivated for 2 h at 56 °C before use. Threefold serial dilutions of sera (40-fold in the initial dilution) in 96-well plates (CoStar, Washington, DC, USA) were incubated with an equal volume of RVFV-SeGFP (100 TCID₅₀ per well) for 1.5 h at 37 °C. Huh7 cells (20,000) were then added to each well, followed by incubation in a 37 °C, 5% CO₂ incubator for 48 h. The eGFP was detected using Cytation1 (Bio-Tek, Winooski, VT, USA). The neutralizing antibody titers were calculated using the Reed–Muench method [24 (link)]. To measure the neutralization activity of each RVFV monoclonal antibody, the isolated monoclonal antibodies were serially diluted in 96-well plates (CoStar, Washington, DC, USA) and incubated with an equal volume of RVFV-SRluc (100 TCID₅₀ per well) for 1.5 h at 37 °C. Then, 20,000 Huh7 cells were added to each well. The cells were incubated in a 37 °C, 5% CO₂ incubator for 24 h. The luciferase activity (Luc) was measured via the Renilla luciferase assay system (Promega, Madison, WI, USA). The neutralization of RVFV (%) was calculated as (1 − Luc_measured/Luc_{virus control}) × 100. Sigmoid neutralizing curves were generated through GraphPad Prism 8 (GraphPad, San Diego, CA, USA).

THP-1 cells were seeded in 96-well plates (Corning Costar) at a density of 100,000 cells per well. First, cells were stimulated for 18 h with rSIP, FLH, LPS, a NOD 1 ligand (positive control for NF-kB; C12-iE-DAP, InvivoGen), and a PRR agonist (positive control for IRF; Poly (dA:dT)/LyoVec™, InvivoGen). The supernatant was then subjected to a colorimetric enzyme assay to measure alkaline phosphatase (AP) activity using the commercial QUANTI-Blue™ solution (InvivoGen). The supernatant was then incubated at 37°C for 3 h, and the optical density was read at 650 nm in an Epoch 2 reader (BioTek). On the other hand, luciferase activity (LUCIA) was measured using the commercial solution QUANTI-Luc ™ (InvivoGen), which has a coelenterazine substrate and stabilizing agents for the luciferase reaction. The light signal produced was then quantified using a Berthold luminometer (Model LB9515), and the signal was expressed as relative light units (RLUs).
Hek-Blue cells (hkb-mtlr4 and hkb-htlr4, InvivoGen) express SEAP under the control of promoters containing binding elements for the NF-κB transcription factor (35 (link)). Hek-Blue cells were seeded in 96-well plates (Corning Costar) at a density of 25,000 cells per well in HEK-Blue™ Detection medium (InvivoGen). Then, the cells were stimulated for 48 h with rSIP, FLH, and LPS, and SEAP was quantified using an Epoch 2 reader (Biotek).

To monitor dose effects, FaDu and Detroit 562 cells were treated with different concentrations of metformin (0, 2, 4, 6, 8, and 10 mM) and loaded into 96-well plates (Corning Costar, Corning, NY, USA) at a density of 2 × 10³ cells/well. After 48 h, 10 µL CCK-8 reagent was added into each well, incubating for 2 h, and then the optical density (OD) value at 450 nm was measured using Multiskan Ascent (Thermo Fisher Scientific). To monitor time effects, FaDu and Detroit 562 cells were treated with 8 mM metformin and loaded into 96-well plates (Corning Costar); 10 µL CCK-8 reagent was added into each well at the indicated time points (0, 12, 24, 36, and 48 h), incubating for another 2 h. The optical density (OD) value at 450 nm was measured using Multiskan Ascent (Thermo Fisher Scientific). This experiment involved three repetitions.