Flow cytometry

HeLa cells (WT and KO) were detached using 20 mM EDTA and resuspended in PBS with 0.5% BSA. The cells were fixed and permeabilized with 4% PFA for 10 min and 0.2% Triton X-100 for 10 min, respectively. After washing with PBS twice, the cells were incubated with the primary antibody for HS (10E4, 1:100) or CS (CS-56, 1:50) (or control without a primary antibody) with rotation for 1.5 h at room temperature. Both primary antibodies and the control group were incubated with goat anti mouse secondary antibodies (TRITC) for 1 h with rotation at room temperature. The cells were sorted with a Sony MA900 Multi-Application Cell Sorter with the following settings: forward scatter (FSC), and side scatter (SSC) signals were collected, and gates were set for single cells. TRITC signals from >4000 events were collected. The data was analyzed with FlowJo software.