Figure 2.

Dispase-Mediated Cell Detachment and Subsequent Floating Culture Lead to Differential Cell Survival via Intercellular Adhesions

(A–L) Treated with Dispase on D13 as indicated in the scheme, the cells with intercellular adhesions lifted up at the edge (A) and then folded up (B–D; sections through the line in C were in Figures 3A and B). EGTA disintegrated the cell sheets (arrow in E). Spheres with a bright ring were visible in 2 days (arrows in D and F). Disintegrated cells (arrowheads in D and F) were either trypan blue positive (arrows in G) or displayed cell blebbing (arrowheads in G), indicative of apoptosis. Serum-mediated reattachment (arrowheads in H) suppressed the apoptosis (H). When the detached cell sheets were dissociated into single cells (arrowhead in I, trypan blue negative) and cultured in suspensions for 2 days, all cells were either trypan blue positive (arrowheads in J) or displayed cell blebbing (inset in J). When the dissociated cells were suspended in Matrigel and cultured as floating clumps, they were trypan blue positive (arrowheads in K). When the dissociated cells were cultured on a Matrigel-coated plate, some cells attached and grew (arrow in L), whereas others remained in suspensions and died (arrowhead in L).

FBS, fetal bovine serum; TE, 0.25% Trypsin/EDTA. Scale bars, 100 μm (A–F, H–K) and 20 μm (G and L). See also Movie S1.