Binding buffer 1x

Calu-6 cells infected or not with the strains (MOI of 5) and PMN treated or not with CM and then infected with H37Rv (MOI of 3) were cultured for further 18 h, detached from the 96-well tissue culture plates, and washed to obtain the pellet. Then Calu-6 and PMN cellular pellets were stained with a fixable viability dye FVD eFluor 780 (eBioscience) and vortexed immediately. After 30 min of incubation at −4°C and protected from light, cells were washed with Binding Buffer 1X (Sigma) and stained with Annexin V kit (Sigma Aldrich) according to the manufacturer instructions. After the time of incubation with Annexin V, cells were washed and fixed with PFA 1% for 20 min and washed with Binding Buffer 1X (Sigma Aldrich). Finally, cells were suspended in flow cytometry buffer and acquired at the cytometer. Results are expressed as percentage of positive or negative cells for Annexin V and FVD eFluor 780.

For both cell lines, HT-29 and ZR-75-1, cells were harvested 7 days after transfection and stained with anti-Annexin V FITC-conjugated antibody (BD Bioscience, 556420) at 20 μl/1 X 10⁶ cells in Binding Buffer 1X (BD Bioscience), for 15 min RT protected from light. Cells were finally resuspended in 400 μl of Binding Buffer 1X and 100 μl of propidium iodide (PI) solution (250 nM) (Sigma-Aldrich) was added to the cells and incubated for 1 min before analysing with the flow cytometer (FACSCalibur, BD). Data was analysed using FlowJo software (FlowJo, BD).