Absorbance plate reader

Cell viability was determined using the Alamar Blue assay (Invitrogen, Thermo Fisher Scientific) and MTT assay (Sigma Aldrich). Briefly, for Alamar Blue assay solution, Alamar Blue dye was diluted in cell culture medium (10% of the total volume in cell culture medium) and added to the 2D or 3D cultures before being incubated at 37 °C for 4 h. After the incubation period, the supernatant from each well was transferred into black well plates. Fluorescence was measured using fluorescence plate reader (Fluoroskan Ascent, Thermo Labsystems, Philadelphia, PA, USA) at Ex/Em 530/620 nm. The Alamar Blue assay allowed the 3D models to be used for obtaining both quantitative and qualitative data since the assay is not toxic and therefore does not kill the cells. For MTT assay, MTT powder was dissolved in cell culture medium (1 mg/mL) and added to the cells. The cells were incubated with MTT solution for 2 h at 37 °C. The MTT solution was then removed from the cells and DMSO was added to the wells. The absorbance was measured at 525 nm using an absorbance plate reader (BioTek, Swindon, UK).

The global DNA methylation status was detected using the MethylFlash Methylated DNA Quantification Kit (EpiGenTek, Farmingdale, NY, USA) according to the manufacturer’s protocol. The amount of DNA used in the assay was 110 ng. Briefly, the H (n = 5) and L (n = 5) cybrids were cultured until confluent and DNA isolated as described earlier. The DNA was bound to strip wells that have a high DNA affinity. The methylated fraction of DNA was detected using capture and detection antibodies and then quantified with an ELISA-like reaction in a microplate spectrophotometer (absorbance 450 nm). The amount of methylated DNA (5-mC%) was proportional to the OD intensity measured with an absorbance plate reader (Bio-Tek, Winooski, VT, USA), calculated according to the kit’s formulas for the relative methylation status of two different DNAs. Samples were run in duplicate, and the experiment was repeated.

Cell growth was tested using WST-1 assay (Abcam, Cambridge, UK) attending the manufacturer’s recommendations at every medium change. Plates were read at 450 nm with a reference wavelength of 680 nm in an absorbance plate reader (Biotek).

The pulmonary production of NO in the BALF was analyzed with a nitrate/nitrite colorimetric assay [33 (link)]. The absorbance was measured at 550 nm using a plate absorbance reader (Bio-Tek Instruments, Inc.).

The pulmonary production of NO in the BALF was analyzed with a nitrate/nitrite colorimetric assay [45 (link)]. The absorbance was measured at 550 nm using a plate absorbance reader (Bio-Tek Instruments, Inc., Winooski, VT, USA).