Annexin 5 kit

CT26 cells were cultured in a 12-well plate at 1 × 10⁵ cell per ml. Cells were either left unpulsed or pulsed with Dox, Cy5-CpG-Chol or a combination for 24 h. After the elapsed time, apoptotic bodies and cells were isolated in accordance with the centrifugation method used by [20 (link)]. In brief, cell supernatant was collected by aspiration and adherent cells were lifted by trypsinisation. The sample was centrifuged at 300 ×g for 15 mins, the resulting pellet was considered the ‘cellular’ fragment, the supernatant was removed and centrifuged at 3000 ×g for 20mins. The pellet of this step was considered to be the ‘cell debris’ containing the sub cellular apoptotic bodies. The two fractions were resuspended in PBS buffer and fractions were assessed for both the presence of Cy5-CpG-Chol and Dox using Flowjo software and presented as contour plots. Annexin V staining of the sub cellular fragment was carried out using an Annexin V kit according to manufacturer's instructions (Biolegend). Gates are drawn on based on unpulsed and/or unstained control, cells whose MFI exceeded the threshold of the gate were considered to be positive for the analysed marker.

Each mouse was given a single intraperitoneal injection. with 1 mg of BrdU (Sigma−Aldrich) and hydrated with BrdU-containing water (0.8 mg/ml) for 5 days. One thymic lobe was digested for TEC, and one was ground for thymocyte analysis. For BrdU staining, freshly isolated thymocytes were incubated with anti-CD4-PE-Cy7, CD8-APC-Cy7, CD25-PerCp, and CD44-APC antibodies. The digested cells were incubated with anti-CD45 PE-Cy7, EpCAM APC, MHCII PE, and UEA-1-Biotin following Avidin-APC-Cy7. The surface-stained cells were then fixed and permeabilized in PBS containing 1% paraformaldehyde plus 0.01% Tween 20 for 48 h at 4°C and then incubated with 2 mg/ml DNase I for 15 min in a 37°C water bath. FITC-anti-BrdU Ab (clone 3D4; BD Pharmingen) was used for BrdU staining according to the manufacturer’s instructions. For Annexin V staining, freshly isolated thymocytes (5 X 10⁵) or total digested thymic cells (1 X 10⁶) were incubated with surface antibodies and then incubated with Annexin V FITC and PI in Annexin V binding buffer. The samples were analyzed within 1 hour following the protocol of the Annexin V kit (Biolegend, San Diego, CA).