Separation and culture of reticulocytes derived in vitro from FVA erythroblasts. (A) Velocity sedimentation profiles of erythroblasts (▪), reticulocytes (○), and extruded nuclei (⬡) derived from the 44-hour cultures of murine proerythroblasts from FVA-infected mice. Fractions collected from the bottom of the gradient were 50 mL for fractions 1 to 9 and 25 mL for the remaining fractions. Fraction 6 represents pooled fractions 1 to 6, and fraction 29 represents pooled fractions 29 to 31. Corresponding sedimentation rates in mm/hour are shown along the top of the figure. Cytospin preparations were stained with 3,3′-dimethoxybenzidine and hematoxylin, and 300 cells were counted for each fraction. Data are ± SEM of 5 separate experiments. (B) Viable cells per mL in cultures initiated with 1 × 10⁶ nascent reticulocytes per mL at 0 hour. Data are ± SEM of 4 separate experiments. (C) Percentage of cells scored as reticulocytes in cultures of nascent reticulocytes. Data are ± SEM of 5 separate experiments. (D) Total RNA per 1 × 10⁸ cells in cultures of nascent reticulocytes. Data are ± SEM of 5 separate experiments. (E) Percentage of biconcave cells in cultures of nascent reticulocytes. Data are ± SEM of 4 separate experiments. (F) ⁵⁹Fe-incorporation into heme (cpm) in cultures of nascent reticulocytes. Data are ± SEM of 3 separate experiments.