Visualizing Mcm2 licensing in intestinal crypts. (A) Representative bright-field images of extracted and unextracted, isolated intestinal crypts. Bars, 100 µm. (B) Representative images of isolated crypts stained with antibodies against Mcm2 (red) or Ki67 (purple). Bars, 10 µm. (C) The Mcm2 labeling index for unextracted and extracted crypts is significantly different (Means ± SEM, n = 10 crypts; t test, P < 0.0001). (D) Representative intestinal crypts stained with Hoechst (blue) and antibodies against Mcm2 (red) and phospho-histone H3 (pH-H3; green). Bars, 10 µm. (E) Representative flow cytometry profiles for extracted and unextracted, isolated, crypt epithelial cells showing Mcm2 versus DNA content. Data are representative of 3 independent experiments. (E’) Suggested model of the licensing profile shown in E. Deeply quiescent cells do not express Mcm2 and have a no detectable Mcm2 signal. Cells expressing soluble Mcm2 (unlicensed G₁) show a similar Mcm2 signal to G₂ cells. After a proliferative-fate decision has been made, origins become licensed and cells commit to S phase entry. Cells enter S phase after maximal origin licensing (active G₁). During the S phase, Mcm proteins are then displaced from DNA during replication. (F) Representative images of extracted and unextracted intestinal organoids stained with an antibody against Mcm2 (red). Bars, 10 µm. (G) The Mcm2-labeling index for unextracted and extracted organoids. Data are displayed as means ± SEM; n = 3 organoids and shows a significant difference (t test, P < 0.0001).