Falcon test tube

Cells were sized and counted with a Coulter Counter (Multisizer 3; Beckman Coulter) using an orifice size of 50 µm and a lower size measurement limit of 1 µm. In addition to the Coulter Counter measurements, cells were alternatively used by flow cytometry with the following protocol. First, cells were resuspended in DMEM with 10% FBS and 1% P/S. The sample was centrifuged at 1,500 rpm for 5 min. The supernatant was removed, and the cells were resuspended in 5 ml of PBS in the tube. Cells were counted using a hemocytometer. We centrifuged 1 ml with 500,000 cells at 1,500 rpm for 5 min. We removed the supernatant and resuspended cells in 500 µl of ice-cold PBS. We added 4.5 ml of ice-cold 70% ethanol in 0.5-ml increments, vortexed in every iteration, and then placed the cells on ice or in a freezer overnight. We centrifuged again at 1,500 rpm for 5 min to remove the supernatant and resuspended the cells in 1 ml PBS. To generate a nuclear signal, we pipetted 10 µl of stock Hoechst 33342 (final Hoechst concentration 100 µg/ml) and incubated the cells for 60 min. Finally, we transferred the mix into a Corning Falcon Test Tube with Cell Strainer Snap Cap (i.e., filter) and measured the fluorescence intensity using a SH800S Cell Sorter.