Effects of CSD and TGFβ on monocyte migration do not involve cell death/apoptosis. (A) Cell death/apoptosis was evaluated by flow cytometry using Molecular Probes kit V13241. Briefly, cells were treated with CSD, TGFβ, or both reagents (see Methods). Cells were then incubated with propidium iodide (PI) and fluorescent annexin V and analyzed by flow cytometry. Gates were set using unstained cells. A typical experiment is shown in which live cells (PI-negative) were gated from dead cells (PI-positive) indicated as Live/Dead. Live cells were further gated into apoptotic cells (annexin V-positive) and non-apoptotic cells (annexin V-negative) indicated as Annexin V: Minus/Plus. To validate the assay, the indicated cells were treated with 20 mM H2O2 (a known inducer of cell death/apoptosis). (B) Apoptosis was evaluated by TUNEL labeling using the In Situ Cell Death Detection Kit, Fluorescein (Product No. 11684795910, Roche, Indianapolis, IN). Briefly, cells on coverslips were treated with CSD, TGFβ, or both reagents (see Methods); fixed; permeabilized; DNA strand breaks fluorescently labeled using TUNEL reagent; and imaged by fluorescent microscopy. A typical experiment is shown in which essentially no cells were labeled except when permeabilized cells were treated with DNase prior to TUNEL labeling. Nuclei were counter-stained using DAPI. (C) Flow cytometry quantification. Average values ± s.e.m. are presented summarizing the results of four independent experiments performed using cells from different subjects. PI + indicates the percentage of dead PI-positive cells; Annexin V + indicates the percentage of the PI-negative cell population that is annexin V-positive (i.e. apoptotic). (D) TUNEL quantification. Three independent experiments were performed using cells from different subjects. The percentage of fluorescent, TUNEL-positive cells was always < 2% except when DNA strand breaks were generated using DNase.