Human annexinv fitc pi apoptosis kit

Following 48 h treatment, the levels of apoptosis were assessed using the “Human AnnexinV-FITC/PI apoptosis” kit (Bender MedSystems, Wien, Austria) as previously described [23 (link)]. Briefly, cell pellets were resuspended in binding buffer and further incubated with Annexin V-FITC and with propidium iodide. Samples were analyzed in a FACSCalibur flow cytometer (BD Biosciences, Erembodegem, Belgium), plotting at least 15,000 events and using the FlowJo 7.6.5 software (Tree Star, Inc., Ashland, OR, USA) [24 (link)].

TUNEL assay was carried out using the ‘‘In situ cell death detection kit—fluorescein’’ (Roche, Boulogne-Billancourt Cedex, France) as previously described [47 (link),48 (link)]. Briefly, cells were fixed in 4% paraformaldehyde (PFA) and cytospins were prepared. Cells were then permeabilized in ice-cold 0.1% Triton X-100 in 0.1% sodium citrate and incubated with TUNEL reaction mixture (enzyme dilution 1:20). Slides were mounted in Vectashield Mounting Media with DAPI (Vector Laboratories Inc., Burlingame, CA, USA), observed in a DM2000 fluorescence microscope (Leica, Wetzlar, Germany) and a semi-quantitative evaluation was performed by counting a minimum of 500 cells per slide. In addition, a specific assay for apoptosis was carried out using the “Human Annexin-V-FITC/PI apoptosis” kit (Bender MedSystems, Vienna, Austria) as previously described [49 (link)]. All flow cytometry analyses were performed using the FACSCalibur flow cytometer (BD Biosciences), plotting at least 10,000 events per sample and using the FlowJo 7.6.5 software (Tree Star, Inc.).