Annexin 5 pe apoptosis detection kit

Cell cycle analysis were performed using propidium iodide staining as previously described^{64 (link)}. Cells were fixed with cold ethanol and resuspended in PBS-citrate Na-BSA containing RNase and propidium iodide. The stained cells were analyzed by flow cytometry (FACSDiva cytometer). Annexin V-PE Apoptosis detection Kit (Immunostep, Salamanca, Spain) was used for the detection of early apoptotic cells. Cells were treated with romidepsin and JQ1 for 24 and 48 h and Annexin V-binding was analyzed by flow cytometry (FACSDiva^TM software, BD biosciences, NJ, USA). The cleavage of poly(ADP-ribose)polymerase-1 (PARP1), indicative of apoptosis, was analyze by immunoblot.
Cell surface markers were analyzed in the Hematology Department of Hospital Marqués de Valdecilla to assess cells differentiation. After 72 h of culture in the presence of 5 nM romidepsin, cell surface markers were analyzed by flow cytometry using the BD FACSDiva flow-cytometer following standard procedures^{65 (link)}. The conjugated antibodies used were: anti-CD20 PE (BD 345793) from BD Biosciences and anti-CD138 FITC (Rafer; Zaragoza, Spain, IQP 153 F).

Cells were analyzed using the Annexin-V-PE Apoptosis Detection kit (Immunostep, Salamanca, Spain), following the manufacturer’s instructions. Splenocytes were resuspended at 1 × 10⁶/ml and surface stained as indicated. The evaluation of apoptosis was based on Annexin-V staining of cells that did not incorporate 7-AAD.