Annexin 5 pi labelling solution

Total mRNA was isolated using the Qiashredder and RNeasy Mini Kit from Qiagen according to the manufacturer's instructions. For cDNA first-strand synthesis, 1 µg of total RNA in 12.5 µl DEPC-H 2 O was mixed with 1 µl of oligo-dT primer (Invitrogen) and heated for 2 min at 70°C. To determine transcript levels of A20, CYLD, Otubain 1, Otubain 2, Cezanne, SHP-1, SHP-2, STAT-1, STAT-3, STAT-5, STAT-6 and GAPDH, the quantitative real-time PCR with the LightCycler System (Roche Diagnostics) face of apoptotic cells was detected with fluorescein isothiocyanate (FITC)-conjugated annexin V binding to PS at the cell surface and necrosis was assessed from the amount of propidium iodide (PI)-positive cells. In brief, 10 5 cells were harvested and washed twice with Annexin washing buffer (AWB). The cell pellet was resuspended in 100 µl of Annexin V/PI labelling solution (eBioscience), and incubated for 15 min at room temperature. After washing with AWB, the cells were analyzed by flow cytometry.