Survival of CLL cells is maintained in high cell density cultures of unsorted PBMCs. (A) The indicated numbers of CLL cells were cultured in 0.5 mL of complete medium per well in 24-well plates, and cell viability was determined after 4 days of culture by flow cytometry after annexin V–PE/7-AAD staining. (B) CLL cells were seeded as described in (A) and cell viability was determined by annexin V–PE/7-AAD staining after 3, 4, 5, and 6 days of culture. (C) PBMCs isolated from CLL samples were enriched for CD19-positive cells by MACS or treated equally without addition of CD19-specific beads. Unsorted and CD19-sorted CLL cells (CD19+) were then cultured under the following 3 conditions: 3 × 105 CLL cells per well in 24-well plates were cultured in 1 mL of complete medium (Control), or in coculture with 3 × 105 preseeded HS-5 cells (Coculture). For high cell density cultures, 3 × 106 CLL cells per well were seeded in 1 mL of complete medium (Cell dense). Cell viability was determined by annexin V–PE/7-AAD staining after 2, 5, and 7 days of culture. Error bars in panels B-C indicate standard deviations of duplicates of one representative example of 3 independently performed experiments.
