Extended Data Fig. 1: CODEX mulitplexed imaging across the healthy human intestine reveals changes in cell composition and organization.

A) Schematic for how CODEX multiplexed imaging was performed on arrays of 4 different sections of either colon and small intestine from the same donor simultaneously. Image processing steps done to extract single-cell spatial data. B) An example CODEX fluorescent image of one region of the small bowel (SB) (1 of 64 tissue sections) for CODEX with 6/54 markers shown for one donor (scale bar = 1 mm and magnified insert = 100 µm) with C) accompanying cell type map following cell segmentation and unsupervised clustering. D-E) Stromal cell type percentages either as a percent of D) All stromal cells, or E) all cells restricted to the Muscularis Externa tissue unit. F) Immune cell type or G) epithelial cell type percentages either as a percent of all cells restricted to the Mucosa tissue unit. H) Percentage of CD57+ Enterocyte cells of all cell types across different areas samples from small intestine to colon. (for D-H: * p value< 0.05, ** p value< 0.01, *** p value < 0.001 by two-sided T test, n=8 donors). (All boxplots in figures are plotted as minimum, 25 percentile, median, 75 percentile, maximum, and outliers as points outside 1.5 the interquartile range). I) Cell map of a representative section (one of 8 donors) of the Duodenum that shows CD57+ Enterocyte presence in glands in the Submucosa where Enterocytes and TA cells are shown in dark grey and Smooth muscle cells in light grey (other cell types not shown) (scale bar = 500 µm). J-K) Quantification of the same-cell density that is measured as an average distance of its 5 nearest same-cell neighbours normalized by the maximal possible same-cell distance within the tissue (n = 64 tissue sections) for J) stromal and K) epithelial cell types. L) A representative cell type map (one of 64 tissue sections from 8 donors) with only plasma cells, CD8+ T cells, and M2 Macrophages shown (scale bar = 500 µm).