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. 2006 Aug 7;203(8):1859–1865. doi: 10.1084/jem.20052136

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Copyright © 2006, The Rockefeller University Press

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Figure 2. — Phenotypic evaluation of tumor colonies of clonogenic assays. (a) Tumor colonies from clonogenic assays were harvested and stained with various antibodies for flow cytometric evaluation. U266 cells grown under different conditions (suspension culture, U266 alone, and with Mo-DCs in clonogenic assays) were analyzed after a 3-wk culture for the expression of cell surface CD138, CD11c, and intracellular Ig light chain. Data shown are gated for the live population. Numbers represent percentages of CD138⁻Igλ⁺ cells. Note that CD11c⁺ DCs are no longer evident at this time point. (b) Phenotype of CD138⁺ tumor cells in clonogenic assays. Cytospins of tumor cells from cultures of tumor cells alone, or tumor–DC cocultures were stained with anti–CD138-PE and Igλ/Igκ-FITC-AlexaFluor488 and analyzed by immunofluorescence microscopy. Red, CD138; green, Ig light chain (κ or λ); blue, DAPI nuclear stain. U266 cells are λ light chain restricted. (c) Histogram of forward scatter (FSC) on flow cytometric evaluation to analyze the size of tumor cells cultured as tumor cells alone or with DCs. (a–c) Results are representative of three separate experiments. (d) Enhanced clonogenicity of cells from tumor–DC cocultures. Tumor cells were harvested from the clonogenic assays of U266 cells originally plated with and without DCs (as in Fig. 1 b) and were serially replated without additional fresh DCs. The numbers of colonies were enumerated microscopically after a further incubation of 3 wk. Results are representative of two separate experiments. Error bars represent SD. iDC, immature DC. *, P < 0.05.