TNF-mediated suppression in vitro is critically dependent on expression of both TNF receptors. (a) In four separate experiments, with two replicates in each experiment, 20,000 whole BM cells from 8–12-wk-old WT, Tnfrsf1-dKO, Tnfrsf1a^−/−, and Tnfrsf1b^−/− mice (all backcrossed for 10 generations with C57BL/6 mice) were plated in methylcellulose supplemented with G-CSF and SCF in the presence and absence of TNF (±TNF). CFU-GM was scored after 7 d in culture. Mean (SD) values for all mice and experiments are shown. (b and c) 100,000 whole BM cells/dish were seeded in semisolid methylcellulose medium supplemented with SCF, erythropoietin, and thrombopoietin, ±TNF, and analyzed for erythroid (BFU-E) and megakaryocyte (CFU-Meg) colony potentials after 8 d in culture. Mean (SD) data from four methylcellulose replicates in two separate experiments are shown. (d) FACS-purified LSK cells from pooled mice of each indicated genotype were single cell sorted into Terasaki plates containing SCF and G-CSF, ±TNF. After 11 d of culture, the number of colonies (>50 cells) was scored. Mean (SD) values from two experiments with two replicate plates each and each plate containing 60 cells are shown.