Zombie yellow

Cell cycling in the HCT116 cells was measured via PI staining of DNA whereby the dividing cells (G2/M) contain double the amount of DNA as those in the growth phases (G0/G1), and therefore are detected by flow cytometry at differing fluorescent intensities. During collection of the PI and zombie yellow (BioLegend, CA, USA) stained cells the living cells (zombie negative) were gated on to remove any false positives in the PI cell cycling plots. Two cell cycling plots were used to detect the shift in population fluorescence, the first being a scatter plot of PI versus forward scatter area to visualise the density and spread of PI-stained cells, and the second being a histogram of cell count versus PI to measure the percent of cells in each phase (Supplementary Figure 12.2). The histogram was then used to analyse the change of cell percentages per cell cycle phase (G0/G1, S or G2/M) in the EG1 treated compared to the control populations. This analysis was done using Kaluza analysis software (version 2.1.1, 2018) by setting gates that encompass the peaks which appear for the G0/G1 phases and S phase; however a defined G2/M phase occurred only at 96 hours. The analysis software was then able to measure the percentage of cells within each of these gates to produce the values which were used to make the cell cycling figure in Rstudio (version 1.4, 2020).

IEL cells were purified using a Percoll gradient as previously described^{41 (link)}. An aliquot was subjected to heating at 65 °C for 20 min to induce cell death. Dead cells were pooled with live cells to serve as an internal control for live/dead staining, which was performed using zombie yellow (Biolegend) as per the manufacturers’ directions. Cells were then washed twice in 2 ml of cold PBS for 5 min, fixed in 4% paraformaldehyde for 20 min at room temperature and pre-treated with anti-Fc antibody for 1 h and subsequently for 2 h with fluorophore-conjugated antibodies. Cells were then washed to remove excess antibody and analyzed by flow cytometry, gating to count live CD45+ cells and analyzed for the abundance of TCR+ , TCR− and sub population of CD8+ cells, F4/80 macrophages or M1/M2 macrophages (CD11c or CD206; see Supplementary Table 2).