Annexin 5 fitc

7-AAD and Annexin-V-FITC were from Cayman Chemical (Ann Arbor, MI, USA). Mouse IFN-γ was from eBiosciences (San Diego, CA, USA). LPS and oligomycin were from Sigma Aldrich (St. Louis, MO, USA). JC-1, MitoSOX and CellROX Green were from Molecular Probes, Life Technologies Corporations (Grand Island, NY, USA). Rabbit polyclonal anti-phospho-p70S6K-T389, laminin, HIF-1alpha, phospho-Akt-S473 and p70S6K were from Cell Signaling Technologies (Danvers, MA, USA). Goat polyclonal anti-Akt and rabbit polyclonal anti-Actin were from Santacruz Biotechnologies (Dallas, TX, USA). Goat anti-Rabbit IgG-IRDye 800CW and Donkey anti-Goat IgG-IRDye 800CW were from LI-COR BioSciences (Lincoln, NE, USA).

The primary and secondary THP-1 macrophage monolayers seeded in either 8-chammber glass slides or 24-well plates and infected with the wild-type, selected MAVA5_06970 gene knockout and complemented clone with MOI of 10:1 as described above, and then processed for apoptosis assays using the TUNEL (Abcam) and Annexin V FITC assay kits (Cayman). For TUNEL assay, infected cells were permeabilized with detergent solution of 0.1% Triton X100 in PBS and 4% FBS for 5 min at room temperature, washed twice with PBS and incubated for 1h at 37C with 50 ul DNA labeling solution consisting of TdT reaction buffer, TdT enzyme, Br-dUTP and double distilled water as per manufactures instructions. Cells were exposed to 100μL of anti-BrdU-Red antibody in rinse buffer and immediately processed for analysis by the fluorescent microscopy.
For the Annexin V-FITC apoptosis assay, primary and secondary THP-1 cell monolayers were infected with either M. avium A5, the MAVA5_06970 knockout mutant or complemented clone for two days, and then according the manufacturer’s protocol (Cayman) incubated with the binding buffer containing the cell-based Annexin V FITC and propidium iodide for one hour at 4°C. Live cells were detached using TrypLE (ThermoFisher), washed with PBS, fixed in 2% paraformaldehyde solution and processed for the flow cytometry as described elsewhere [48].