FIGURE 1.

Quantification of total cell numbers, the number of BrdU+ cells and the number of myosin+ cells when C2 myoblasts are maintained in 2% FCS ±PDGF. ‘Lower density’ cultures (lower panels) were initiated at 2.4 × 10⁴ cells/plate and ‘higher density’ cultures (upper panels) were initiated at 4 fold higher density. Cultures were initiated in the serum-rich, proliferation medium and 24 hours later switched into medium consisting of 2% FCS ±PDGF which was replaced daily; BrdU was administrated to the culture medium during the last 2 hours before fixation. Cultures were doubly labeled with antibodies against BrdU and myosin. Total cell numbers reflect the total number of nuclei including nuclei in myotubes (when myotubes are present). The number of myosin+ cells also reflects the number of nuclei in both mononucleated cells and myotubes. The height of the y-axis used for presenting the number of myosin+ cells in the higher and the lower density cultures was chosen in order to maintain the same ratio used for graphing the total cell numbers. Each analysis is based on the average of two parallel plates analyzing five microscopic fields per plate using a 25x objective. Note that in all other experiments we used a 40x objective counting at least ten microscopic fields; the area of one microspic field observed with the 25x objectives is about 2.5 fold larger than the area of a field observed with a 40x objective.