Quantifying RL2 Internalization and Cell Death

Analysis of RL2 internalization and cell death induction was performed with FlowSight^® Imaging Flow Cytometer (Amnis/Merck Millipore, Darmstadt, Germany). MDA-MB-231 or MCF-7 cells were treated with RL2 or Rhodamine-labelled RL2 [8 (link)] for measuring cell death or internalization, respectively. Samples for measuring cell death were additionally stained with propidium iodide (PI). The samples were excited with a 488 nm laser. Emission was detected in channel 4 and bright field images were acquired in channels 1 and 9. For every sample, 10,000 events were recorded. Data were analyzed with IDEAS software version 6.2 (Amnis/Merck Millipore, Darmstadt, Germany). For internalization, Rhodamine-positive cells were taken as RL2-positive cells. RL2-induced cell death was calculated via the percentage of PI positive cells.