Ldh detection kit

Cell viability was assessed using the lactate dehydrogenase (LDH) detection kit (Sigma-Aldrich). To avoid potential interference of bacteria with LDH activity, the intracellular LDH assay was applied (32 (link)). Briefly, 3D A549 cells were washed twice with Hank’s Balanced Salt Solution (HBSS, Life Technologies) and lysed with 1% Triton X-100 (Sigma-Aldrich) by vigorous pipetting. LDH activity was further determined as described in the manufacturer’s protocol. Afterwards, cell viability was expressed as a percentage of the uninfected control. When viability was lower than 80% and a statistically significant difference was obtained compared to the uninfected control (p< 0.05), the commensal bacterium was considered as cytotoxic (33 (link), 34 (link)).

The cytotoxic effect of CANE was first determined by MTT assay. Briefly, 5 × 10⁴ A549 cells/well were incubated in the presence of CANE at the concentrations of 25, 50, 100, and 150 μg/ml for 24 h at 37 °C in a CO₂ incubator. After incubation, cells were treated with MTT solution (5 mg/ml) to produce dark blue colored formazan crystals, which were further dissolved in 50 μl of DMSO. Finally, absorbance was measured at 540 nm in a microplate reader (Bio-Tek instrument Co., WA, USA).
Cytotoxicity was also determined by the lactate dehydrogenase (LDH) assay. For the LDH assay, cells were treated with various concentrations of CANE, as mentioned above, for 24 h at 37 °C. After incubation, media were removed and processed to evaluate extracellular LDH release using an LDH detection kit (Sigma-Aldrich, St.Louis, USA) as per the manufacturer’s guidelines. Finally, cytotoxicity was evaluated by calculating absorbance at 490 and 690 nm.
Furthermore, microscopic examination was done to determine morphological changes in A549 cells after exposure to CANE (25, 50 and 100 μg/ml) using an inverted microscope (Nikon Eclipse TS200, Nikon Corp., Tokyo, Japan). In addition, cytotoxic potential of CANE against PC-9 cells was evaluated by MTT, LDH assay as mentioned earlier along with the western blotting of the apoptotic proteins.

Membrane integrity was assessed by measuring the amount of cytoplasmic lactate dehydrogenase (LDH) released into the medium, using an LDH detection kit (Sigma-Aldrich, USA) following the manufacturer’s protocol. The absorbance was read from the top of the well using the SpectraMax iD3 microplate reader (Molecular Devices, USA). LDH activity levels are presented as a percentage of the activity in the SCS no treatment group.

After infection, cell supernatant was collected and centrifuged (3,700 rpm, 15 min). LDH activity of the resulting supernatant, a direct measurement of the dead fraction of epithelial cells, was determined using the LDH detection kit (Sigma-Aldrich) following the manufacturer's instructions. Cytotoxicity was expressed as a percentage of an uninfected control treated with 1% triton X-100 (Sigma-Aldrich). Efficient lysis of cells was achieved through vigorous pipetting.
After removing the supernatant, cell monolayers were rinsed once with Hank's Balanced Salt Solution (HBSS, Life Technologies). For fluorescence microscopy, monolayer cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 20 min and stained with 4′,6-Diamidino-2-Phenylindole (DAPI, Life Technologies) to visualize epithelial cell nuclei. Afterwards, three representative images were taken per well using an EVOS FL Auto Imaging System (Life Technologies) equipped with a 20x objective (final magnification: 368x). The number of adhering cells in each image was determined by enumerating epithelial cell nuclei using the image processing application ImageJ (LOCI, University of Wisconsin). For calculation of cell adherence, the average cell number of three images was calculated and compared to the uninfected control. Cell detachment was then calculated as the complement of the adherent fraction.