Table 1

In Vitro Hematopoiesis and Differentiation of Wild-type and C/EBPα−/− Cells

CSF
ColoniesSegmented neutrophils
Wild-type−/−
% wild-type
IL-6134 ± 252.0 ± 1.4<1
IL-6 + soluble IL-6 receptor200 ± 49 26 ± 113.4 ± 1.8
IL-6 + soluble IL-6 receptor + G-CSF266 ± 50 82 ± 4012 ± 4.8
G-CSF150 ± 700.5 ± 1<1
G-CSF + G-CSF receptor148 ± 66 63 ± 2866 ± 26
IL-6 + soluble IL-6 receptor + G-CSF227 ± 58 37 ± 1139 ± 23
IL-6 + soluble IL-6 receptor + G-CSF + G-CSF receptor retrovirus192 ± 23 46 ± 1184 ± 12
IL-3249 ± 25217 ± 8635 ± 18
GM-CSF181 ± 81163 ± 5740 ± 5.7
  • Fetal liver cells (105 cells for CFU-G and CFU-IL6, and 2 × 104 cells for CFU-IL3 and CFU-GM) were plated in methylcellulose medium containing the indicated cytokines. The concentration of cytokines is as described in Materials and Methods. Colonies containing >50 cells were counted after 6–7 d. Three to five animals of each genotype were analyzed. Wild-type animals (+/+) gave a result similar to that obtained from heterozygous animals (+/−). The number of CFU-IL6 from C/EBPα−/− livers was derived from only three colonies in one plate, which morphologically showed all macrophages after cytocentrifugation of the colonies onto slides and staining with Wright-Giemsa. Differential counts were performed as follows: two to four colonies were pooled from each plate on two successive days and cytocentrifuged onto glass slides. The slides were stained with Diff-Quik solution. The percentages of segmented granulocytes were determined by counting 200–300 cells from each slide, and calculating the percentage of granulocytes in knockout colonies compared with that in wild-type. Each number was derived from 3 to 4 individual animals and a total of at least 16 colonies for CFU-G and CFU-IL6.