Characterization of early primary cell preparations. (A) EdU incorporation after a 50 μm five hour pulse of primary cell aggregates (two days-old). (B) Semi-quantitative RT-PCR of em-alp-1 and em-alp-2 genes with serial ten-fold dilutions of cDNA from primary cells (PC) and germinal layer (GL). The experiment was repeated three times with similar results. (C and D) Representative microscopy fields of cell suspensions obtained from the germinal layer (C) and from primary cells (D). EdU + and EdU − germinative cells are indicated by filled and open arrowheads, respectively. The inset in D shows a close-up of two EdU + germinative cells in the primary cell preparations. (E and F) Transmission electron microscopy (TEM) of primary cell aggregates (two days-old). (E) Accumulation of germinative cells. (F) A muscle cell (m), containing myofibers and extensive smooth endoplasmic reticulum, and a germinative cell (g) in the periphery of an aggregate. Notice also cell debris surrounding the cells. Bars represent 4 μm (A, E, F and inset in D) and 20 μm (C and D).