Propidium iodide staining assays for cell proliferation and cell death quantifications

Cells were seeded into 96 well plates at 500 cells/well and were treated with purified 2XMBP-scFv proteins for 72 hours. The media containing purified 2XMBP-scFv proteins was removed and replaced with media containing PI/RNase staining buffer (BD Biosciences) and Hoechst dye (Thermo Fisher). The plates were imaged and the nuclei were quantified using a Cytation3 Cell Imaging Multi-Mode Reader (BioTek). The media was then replaced with fresh media and cells were cultured for another 96 hours. Media was then replaced with media containing PI/RNase staining buffer (BD Biosciences) and Hoechst dye (Thermo Fisher). The plates were imaged again and the nuclei were quantified using a Cytation3 Cell Imaging Multi-Mode Reader (BioTek). Total cell number was quantified based on Hoechst staining. Percent cell death was quantified from the PI positive cell number divided by total cell number. Experiments were performed at least three times for biological replicates. Statistical significance was determined using the unpaired t test (GraphPad Prism).