Annexin 5 staining buffer

After treatment as above, 0.6 × 10⁶ PBMC per tube were centrifuged at 400 × g for 6 min and the pellet was resuspended in 100 µL of Annexin V staining buffer (BioLegend Cat # 422201, San Diego CA) or 100 µL of HBSS (for lactadherin only samples) containing 5 µL of Human TruStain FcX™ (BioLegend Cat # 422302). After a 5-min room temperature incubation in the dark, the cells were then stained with 2.5 µL of Pacific Blue™ anti-human CD45 Antibody (BioLegend Cat#304029 RRID : AB_2174123, San Diego, CA) and 5 µL of Alexa Fluor® 647 Annexin V (BioLegend Cat # 640912, San Diego, CA) with and without 1 µL of FITC-bovine lactadherin (Prolytix, Essence Junction, VT) for 15 min at room temperature in the dark. The addition of 5 µL of 7AAD for a further 5 min in the dark was used to discriminate live from dead cells. Prior to analysis, the samples were diluted with 100 µL of HBSS (Lactadherin only) or 100 µL of Annexin V buffer (Annexin V ± lactadherin samples) and then analyzed on a BD FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ). Alternatively, doubly labeled cells were analyzed on an AMNIS imaging flow cytometer (Luminex, Austin, TX). Data are expressed as percent of parent.

Breast cancer cell lines were treated and cultured as described for Trypan Blue studies. At 72 hours posttreatment, cells were harvested by scraping and washed with Annexin V staining buffer (BioLegend, San Diego, CA). Cells were then stained with fluorochrome-labeled Annexin V (3 μL added in 40 μL total volume) (BioLegend) at 4°C for 15 minutes, followed by the addition of propidium iodide (PI) (1 μg/mL) (Sigma-Aldrich) immediately prior to analysis using a FlowSight imaging flow cytometer (employing the 642 nm and 488 nm lasers) and running IDEAS version 6.2 software as described previously [12 (link), 13 (link)].