Guava personal cytometer

At various time points after treatment with XN, DXN, or TXN, adherent and floating cells were collected and fixed in 70% ethanol at one million cells per aliquot. Samples were centrifuged at 500× g, washed with PBS, and re-suspended in cellular DNA staining solution containing 40 mg/mL propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) and 100 mg/mL RNase A (Sigma-Aldrich) in PBS. After a 15-min incubation at room temperature, cell populations were quantified using a Guava personal cytometer (Guava Technologies).

At various times after treatment with XN, DXN, or TXN, adherent and floating cells were collected for analysis. Apoptosis was assessed using flow cytometry-based annexin V (Invitrogen™. eBioscience™ Annexin V Apoptosis Detection Kit PE, Thermo Fisher, Waltham, MA, USA) and a multicaspase assay kit (MilliporeSigma, Burlington, MA, USA). The annexin V assay is based on measurement of externalization of phosphatidyl serine, a common characteristic of cells undergoing apoptosis. The multicaspase assay is based on measurement of caspase enzymes activated during apoptosis. Cells were trypsinized, washed in Dulbecco’s Phosphate Buffered Saline (D-PBS), and stained with Annexin V, sulforhodamine-valyl-alanyl-aspartyl-fluoromethyl-ketone (SR-VAD-FMK), and 7-amino-actinomycin D (7AAD) according to the manufacturer’s instructions. Cell populations were quantified using a Guava personal cytometer (Guava Technologies, Burlingame, CA, USA).