Anti phosphatedylserine

3x10⁴ cells were cultured on coverslips, treated with HT (10-100 μM), cetuximab (1-10 μg/ml) with/without EGF (5 ng/ml) for 8-24 or 48 h (with treatment of HT every 24 h) and then fixed in paraformaldehyde (4%, 10 min), washed in PBS and permeabilized, if necessary, with Triton X-100 (0.5% in PBS containing 0.5% BSA) and then incubated with BSA (45 min). Cells were then incubated for 16 h with anti-EGFR (1:40), anti-AIF (1:50, Cell Signaling), anti-phosphatedylserine (1:50, 05-719 Merk Millipore), anti-occludin (1:80, 71-1500 ZYMED) diluted in PBS containing 0.5% BSA. To investigate phosphatedylserine externalization on cell membranes, cells were fixed in paraformaldehyde (4%, 10 min), washed in PBS, incubated with BSA (45 min) without permeabilization, and then incubate for 16 h with anti-phosphatedylserine (1:50, 05-719 Merk Millipore) diluted in PBS containing 0.5% BSA. After incubation with the secondary antibody ALEXAFLUOR 488 anti-mouse or anti-rabbit (A11001, A11011 respectively; 1:200 1 h, Thermo Fisher), cells were washed and incubated with DAPI (1 μg/mL, 20 min), washed and the coverslips were mounted in Fluoromount Aqueous Mounting Medium.