Accuri c6

To analyze apoptosis, cells were treated as indicated and allowed to recover for 4 days. Floating cells were collected, and then attached cells were collected with trypsin and combined. After centrifugation and washing, the cells were incubated with Alexa Flour 488 annexin V and 1 µg ml^–1 PI in 1× annexin-binding buffer for 15 min in the dark (Thermo). After resuspending in additional binding buffer, the cells were analyzed on an Accuri C6 (Beckman) using FL1 and FL3.
For cell cycle analysis, 23 h after treatment, cells were pulsed with 20 µM EdU and incubated for an additional hour (Thermo). Cells were collected with trypsin, washed with 1% BSA in PBS and then fixed with Click-IT fixative D. After washing with 1% BSA in PBS, the cells were permeabilized with 1× component E for 15 min, before performing Click chemistry with Alexa Flour 488 azide for 30 min in the dark. Cells were washed with 1× component E, and then resuspended in 500 µl FxCycle PI/RNase (Thermo) for 15 min before analyzing on Accuri C6. Standard gating for cells versus debris and singlet was conducted.

Bacterial cell densities were measured using an AccuriC6 flow cytometer and intact cells were distinguished from damaged cells using SYBR Green (SG; Thermo Fisher)/propidium iodide (PI; Sigma) staining, as previously described (79 (link)). During coculture, bacterial cells were recovered from the cell layer by gently pipetting up and down the apical washing fluid (PBS with calcium and magnesium ions), and the bacterial densities in the apical washing fluid were determined by using flow cytometry.