Annexin 5 fitc and propidium iodide

Apoptosis was assessed by Annexin V/propidium iodide (PI) flow cytometry assay following an incubation of 16 hours with indicated drugs. Cells were then trypsinized and collected, washed with PBS and resuspended in binding buffer. Cells were stained with Annexin-V-FITC and propidium iodide (BD Biosciences, San Jose, CA, USA, 556547) and flow cytometry was undertaken on a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA).
The PROTEOSTAT® aggresome detection kit (Enzo Lifesciences, Farmingdale, NY, USA, ENZ-51035-0025) was used to detect aggresome formation following drug treatment at 4 and 18 hours. Following indicated treatment, cells were trypsinized, washed twice with PBS, fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100. Cells were then stained with the PROTEOSTAT® Aggresome Red Detection Reagent and analyzed by flow cytometry on the FL3 channel of the FACSCanto II.

For viability assay, THP-1 cells at a concentration of 0.5 x 10⁶/ml were treated with different doses of B. abortus RNA in the presence of IFN-γ for 48 h. THP-1 cells treated with 2% paraformaldehyde (PFA) were also included as positive control. After 48 h, cells were washed and incubated with Annexin V-FITC and Propidium Iodide (BD Biosciences) for 10 min on ice in darkness. Then, cells were evaluated in the quadrants of Annexin V⁺/PI^- (early apoptosis), Annexin V⁺/PI⁺ (late apoptosis) and Annexin V^-/PI⁺ (necrosis). After labelling, cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences) and data were processed using CellQuest software (BD Biosciences).

Cardiomyocytes were stained with Annexin V-FITC and propidium iodide (BD Pharmingen, USA), and then subjected to flow cytometry to determine apoptosis. The apoptotic rate was calculated as the percentage of Annexin V-positive and propidium iodide-negative cells divided by the total number of cells in the gated region.