Examination of SCCM. (A) Measurement of IL-7 secreted by stromal cells from young and old mice. A bioassay for IL-7 was used to measure the amount of IL-7 secreted by FACS-purified stromal cells in 72 hours. SCCM was used at a final concentration of 10%. The results shown are the average ± SD of 3 to 4 replicate wells using the pre-B–cell line BC715; similar results were found using two other pre-B–cell lines, BC76 and 2E8. Similar results were also obtained when 20% and 30% SCCM were used and with SCCM from 4 separate collections (data not shown). The responses to 0.001 and 0.0025 ng/mL rIL-7 are shown for comparison. (B) Production of secreted M-CSF activity by stromal cells. The amount of M-CSF activity present in SCCM from FACS-purified stromal cells was determined by a CFU-M assay. The number of macrophage colonies having greater than 20 cells was determined using a dissecting microscope after culture in semisoft agar for 7 days. The data are reported as the average ± SD of 3 to 4 plates per group. The results shown are from one representative experiment of three performed with different collections of SCCM after 72 hours. The results shown used SCCM at 20%; no age-related differences were observed when the SCCM was used at 10% (data not shown). The responses to 2.5 and 7.5 U/mL rM-CSF are shown for comparison.