Figure 13.
Effect of ALL3 HDSN on proliferation of LD ALL3 cells. A. Pictures of LD and HD ALL3 cells on day 2 that were stained with the MIB-1 Ki67 antibody and counter stained with nuclei stain DAPI. The LD cells hadn’t grown at all by day 2, their viability was only 50%, but >90% of the viable cells were Ki67+. The HD cells had grown to 4.85 × 105 cells by day 2, were 84% viable, and 100% were Ki67+. B. Bar graph of the HD and LD ALL3 cells started on day 0 at 20 × 104 and 0.5 × 104 cells/ml and cells were stimulated using HDSN were pulse labeled with EdU at days 1, 2, 4, and 6. Percent of EdU+ cells are shown on each day. C. Representative pictures of the HD and LD ALL3 cells started on day 0 at 2 × 105 and 5 × 103 cells/ml that were pulse labeled with EdU at days 1 and 6. Serial pulse labeling with EdU for ALL3 cells growing for 6 days in ALL3 media at both HD of 2 × 105 and LD of 5 × 103 cells/ml, the latter with and without addition of HDSN from another culture of ALL3 cells growing exponentially for 3 days starting at 3 × 105 cells/ml. Despite their poor growth, the LD cells alone initially had a higher percentage of cells (41%) incorporating EdU (middle panel) than the proliferating HD cells (left panel; 28%), but the poorly growing LD cells in S phase dropped to 26% by 6 days (middle panel) whereas the percentage of LD cells in S phase stimulated to grow by the HDSN (right panel) declined less rapidly (39% to 30%). The cell counts/ml and percent EdU + cells are shown on each day. The lower percent in S phase of the HD cells at 6 days (18%) is probably because the HD ALL3 cells were entering saturation phase. The higher percentage of LD ALL3 cells in S phase than the HD cells on day 1 (41% vs 28%) was because the LD ALL3 cells were prepared by dilution of proliferating HD ALL3 cells were still mostly viable by day 1 and some continued to enter S phase which they were unable to complete before dying (middle panel). The LD cells rescued by HDSN (right panel) were growing nicely by day 6 and had about the same percentage of cells in S phase (30%) as the HD cells on day 1 (28%).