Live dead green dead cell stain kit

CD4+ cells were isolated from bulk PBMCs using the CD4+ T Cell Isolation Kit with MicroBeads for column-based magnetic activated cell sorting (MACS)(Miltenyi Biotech, CAT# 130-096-533). MACS was performed according to the manufacturers protocol. Cells were stained with PE/Cy7-CD25 (BioLegend, M-A251, CAT# 356108, RRID: AB_2561975), PE-CD45RA (BioLegend, HI100, CAT# 304108, RRID: AB_314412), PerCP/Cy5.5-CD45RO (BioLegend, UCHL1, CAT# 304222, RRID: AB_2174124), BUV395-CD3 (BD Biosciences, UCHT1, CAT# 563548, RRID: AB_2744387), and LIVE/DEAD Green Dead Cell Stain Kit (ThermoFisher Scientific, CAT# L23101). Pooled cells from all groups were used to generate the LIVE/DEAD single-color control, all fluorescence minus one (FMO) controls, and unstained controls. Ultracomp eBeads (ThermoFisher Scientific, CAT# 01-2222-41) were stained to generate single color controls. Analysis was performed using FlowJo (Version 10, RRID: SCR_008520). FACs gating strategy is detailed in Supplemental Figure 1.

Human gingival epithelium progenitor cells stimulated by P.ginigvalis at different MOIs and P.ginigvalis-challenged cells used in epithelial barrier function as mentioned above were collected and washed with PBS. After washing, cells were immediately stained with a Live/ Dead Green Dead Cell Stain Kit (Thermo Fisher Scientific) for 30 min at 4°C in dark. Then, fluorescence signals were read by a BD LSR II with Violet flow cytometer (Becton Dickinson). Heat-killed cells were included as a positive control for complete cell death.