Elx808iu microplate reader

After isolation, neutrophils were suspended in HBSS buffer containing 80 μM ferricytochrome C type III (from bovine heart, C-7752, Sigma-Aldrich), 0.5 mM CaCl₂, and 1 mg/mL glucose, and then distributed in a 96-well plate (1 × 10⁵ cells/100 μL/well) to be incubated for 10 min at 37 °C before stimulation. Plates were then incubated at 37 °C in an automated ELx808IU microplate reader (BioTek, Winooski, VT, USA) to record cytochrome C reduction, measuring at intervals of 5 min for 90 min the Δ O.D. 550 nm/465 nm. O₂⁻ production was finally calculated using an extinction coefficient of 24.5 mM.

BALF was collected according to the routine bronchoscope operating procedure: 20 mL of normal sterile saline, warmed to body temperature (37°C), was injected for bronchoalveolar lavage. BALF volume of 5–10 mL was recovered and sent for microbial inspection in a disposable sterile silicon plastic bottle. Microbial inspection was performed using an ELx808IU microplate reader (BIoTek, Winooski, VT, United States). The recovered BALF was transferred to a 10-mL centrifuge tube using a disposable sterile pipette. After centrifugation at 1,000 rpm for 10 min using a microfuge 20R centrifuge (Beckman Coulter, Brea, CA, United States), 600–800 μL of supernatant was collected and stored at –40°C for further tests. Venous blood (3 mL) was collected and centrifuged at 3,500 rpm for 10 min; serum was collected and stored at 4°C.
The GM test was performed to detect GM levels in serum and BALF samples using the one-step enzyme immunoassay sandwich method (Aspergillus antigen detection kit). The BDG test was mainly performed to detect BDG in serum and BALF samples using the dynamic turbidimetric method (fungal dextran detection kit). All tests were conducted following the manufacturer’s instructions.
Serum GM > 0.5, BALF GM > 0.7, serum BDG > 100 pg/mL, and BALF BDG > 200 pg/mL were defined as positive values for fungal infection (9 (link), 20 (link)).

Cell viability and proliferation were determined using MTT, MTS, and colony formation assays were performed as described in our previous reports (17 (link)). Briefly, for MTT assay, cells were incubated in 96-well plates and exposed to the indicated concentration of 40, 60, 90 nm GB-AgNPs or GB-extracts or AgNO₃ or no-treatment control for 24 h. 10 µl MTT (5 mg/ml) was added to each well and incubated for 4 h. The medium was replaced with 150 µl of DMSO to dissolve the crystal formazan dye and absorbance was detected at 540 nm using an ELX808IU Microplate Reader (BioTek, USA). For MTS assay, cells were plated in 96-well plates and exposed to the indicated concentrations of 40 nm GB-AgNPs or no-treatment control for 12, 24, and 36 h. 20 µl of MTS was added to each well and incubated for 1 h, after which the absorbance of the MTS signal was calculated after absorbance detection at 490 nm. For colony formation assay, cells were treated with the indicated amount of 40 nm GB-AgNPs or no-treatment control for 24 h and cultured at a density of 500 cells/well in 6-well plates. Then the medium was changed every 3 d, and after two weeks cell colonies were stained with Giemsa stain solution (Solarbio, CN). Visible colonies were photographed and counted using a Gel DocTMXR⁺ Molecular Imager system (BioRad, USA).

The PC9 and NCI-H460 cells were seeded into 96-well plates in 1 × 10⁵ cells/well, incubated for 24 h and then exposed to the different fractions of F. luteovirens or negative control for 24 h. Cell viabilities were evaluated with an MTT assay (Sigma, St. Louis, MO, USA). In brief, 10 μL of 5 mg/mL MTT was added to each well and incubated for 4 h. The medium was replaced with 150 μL of DMSO to dissolve the crystal formazan dye, and absorbance was detected at 490 nm using an ELX808IU Microplate Reader (Bio-Tek, Winooski, VT, USA). The inhibition rates were calculated as follows: inhibition rate (%) = (average A₄₉₀ in the control group−average A₄₉₀ in the experimental group)/(average A₄₉₀ in the control group−average A₄₉₀ in the blank group) × 100%. The IC₅₀ value was determined as the concentration that caused 50% inhibition of cell proliferation.

Cell viability was evaluated by the MTT test (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) (Merck Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. 15 × 10⁴ cells were seeded on 96-well plates and incubated to reach 90% confluence. Next, cells were treated with experimental medium containing 0.0001–40 μM DON for 24 h. Four hours prior to the end of the incubation period, 5 mg/mL MTT solution was diluted to a final concentration of 0.5 mg/mL in each well. The formazan crystals formed by viable cells were dissolved in 10% SDS with 0.01 M HCl (100 μL), incubated overnight and measured using an ELX 808IU microplate reader (BioTek, Winooski, VT, USA) at 570 nm. The results are expressed as a percentage of non-treated cells. Each experiment was conducted in six replications.

Cells were incubated in 96-well plates and exposed to the indicated concentration of PAC (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 μM) or vehicle control (DMSO) for 24 h. Cell viabilities were evaluated with an MTT assay (Sigma, USA). In brief, 10 μL of 5 mg/mL MTT was added to each well and incubated for 4 h. The medium was replaced with 150 μL of DMSO to dissolve the crystal formazan dye and absorbance was detected at 540 nm using an ELX808IU Microplate Reader (BioTek, USA).
Cells were seeded in 96-well plates and exposed to the indicated concentration of PAC (0.1, 0.2, 0.3, 0.4, 0.6 μM) or vehicle control (DMSO) for 24 h. Cell proliferation was measured with a BrdU assay (Abcam UK): BrdU (10 μM) was added to each well and incubated for 12 h, and the BrdU signal was calculated after absorbance detection at 450 nm.
Cells were plated in 96-well plates and exposed to the indicated concentrations of PAC (0.2, 0.4, 0.6 μM) or vehicle control (DMSO) for 12, 24, and 36 h. Cell viability was examined with an MTS assay (Sigma, USA), 20 μL of MTS was added to each well and incubated for 1 h, after which the absorbance of the MTS signal was calculated after absorbance detection at 490 nm.