Nanoquant infinite m200pro multiplate reader

Cytotoxicity was evaluated as cell viability by the MTT assay using two different human cells lines: the adenocarcinomatous human alveolar basal epithelial cells A-549 and the human lymphoid MEC-1 cells (DSMZ, ACC 497). A-549 and MEC-1 cells grew in Dulbecco’s MEM (Sigma, Milan, Italy) added in 10% (v/v) FBS (EuroClone, Milan, Italy), 100 U/mL penicillin/streptomycin (Sigma-Aldrich, MO, USA), and 2 mM L-glutamine (EuroClone, Milan, Italy) at 37 °C in 5% (v/v) CO₂. Then, 20,000 cells/50 μL were seeded in each well of a 96-well flat-bottom microtiter plate (Sarstedt, Milan, Italy) and incubated overnight at 37 °C in 5% CO₂. Serial twofold dilutions of D-BMAP18, Pro-D-BMAP18, and Pro-D-BMAP18 in the presence of elastase (molar ratio, 100:1) were prepared in the same cell growth medium, and 50 μL of the samples were added to the cells. After 20 h of incubation at 37 °C under 5% (v/v) CO₂, 20 μL of MTT (5 mg/mL in PBS) was added to each well. After 4 h incubation at 37 °C in the dark, 100 μL of 10% (w/v) Igepal (Sigma-Aldrich, MO, USA) in 0.01N HCl was added to each well and the plate was incubated overnight at 37 °C under 5% (v/v) CO₂. Cell viability was evaluated as absorbance at 570 nm using the Nanoquant infinite M200pro multiplate reader (Tecan, Mannedorf, Switzerland).

The
determination of specific immunoglobulins (Igs) against fecal bacteria
was as previously described,^{33 (link)} with the
following modifications. Removal of debris/rat cells was performed
via filtration through a 100 μm strainer and centrifugation
at 400g for 10 min (4 °C). After being washed
and heat-killed, bacteria were resuspended in 20 mL of PBS, and 100
μL of this suspension was used for overnight coating (4 °C)
in a 96-well ELISA plate. The number of bacteria in each suspension
was measured by spectrophotometry (600 nm) and adjusted across experimental
groups. Blocking was carried out as already described.^{33 (link)} Rat sera were diluted 1:100 and incubated overnight
at 4 °C for detection of IgG, IgM, and IgA. Incubation with secondary
antirat IgG, IgM, and IgA (Bionova, Spain) (1:10,000) for 1.5h in
darkness was followed by the addition of the HRP substrate (Sigma-Aldrich,
USA). Absorbance was measured using a Nanoquant Infinite M200 Pro
multiplate reader (Tecan, Switzerland).