Pe conjugated annexin 5 antibody

The isolation of bulk tumor cells as well as residual cancer cells was performed prior to or two to four weeks after treatment with Dox and downregulation of the oncogenic driver. This extended period, which exceeded the time required for a macroscopic regression of the tumors by more than 7 to 21 days (Lin et al., 2013 (link)), was chosen to ensure that the cancers had completely regressed and only dormant cancer cells were analyzed. GFP-positive bulk tumors or areas with small residual tumor masses that contained GFP-positive cells were isolated from the pancreata under a fluorescent stereoscope. To determine the number of GFP-positive apoptotic cells using flow cytometry, excised tissues were processed for enzymatic dissociation and biotinylated anti-CD31 and anti-CD45 antibodies (BD Pharmingen) were used for elimination of endothelial and hematopoietic lineages using AutoMACS Pro (Miltenyi Biotec) as described previously (Lin et al., 2013 (link)). A PE-conjugated Annexin V antibody (BD Pharmingen) was used for staining of apoptotic cells. The flow cytometric data was acquired on a BD FACSCalibur at the UNMC Cell Analysis Core Facility. Data was analyzed using the FlowJo V9.8 software (Tree Star Inc.).

0.75 million cells were plated on P60 dishes. The next day, cells were washed once with PBS and incubated in either low glucose (2.5mM) or high glucose (25mM) containing media (DMEM containing 10% dialyzed FBS) for 36-48 hours, or no glucose (0mM) or high glucose (25mM) for 24 hours. Cells were collected at the indicated time point, washed once in PBS, trypsinized, and pelleted. Cells were then washed in 1X AnnexinV buffer and stained with AnnexinV and 7-amino-actinomycin D (7-AAD) according to the manufacturer’s protocol (BD Pharmingen, San Diego, CA). Briefly, cells were re-suspended in AnnexinV buffer to a concentration of 1×10⁶/mL. Cells were then stained with 5 μL of phycoerythrin (PE)-conjugated AnnexinV antibody (Cat #556422 BD Pharmingen) and 5 μL of 7AAD (Cat# 51-2359KC BD Pharmingen) and incubated at room temperature for 15 minutes. 400 μL of AnnexinV buffer was then added to each sample with gentle mixing. Stained cells were analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and flow cytometry data was analyzed using FlowJo v. 10.1 software (Tree Star Inc., Ashland, OR).