Multiplex analysis of cytokines.

<p><i>A, In vitro cytokine production by CSCs and parental human tumor H460 cells.</i> Cells were cultivated in 96-well plates for 24 h in complete RPMI 1640 medium; samples of conditioned media were collected. Cells were fixed, stained with Hoechst 33342, and cell numbers were determined using image cytometry. Concentrations of human cytokines, chemokines, growth factors, MMPs, adhesion molecules and cancer antigens were analyzed using Luminex technology. Concentrations of cytokines pg/10⁶ cells/ml were calculated. Only factors with significant differences in their concentrations are presented. B, <i>Analysis of murine cytokines in extracts of xenografted parental H460 and CSCs-derived tumors.</i> SCID mice were inoculated s.c with 5×10⁵ of parental H460 or CSCs (5 mice per group). Samples of tumors, derived from parental H460 cells and CSCs, were sonicated, and concentrations of 19 murine cytokines in cellular extracts were measured using multiplexed cytokine assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003077#s2" target="_blank">Materials and Methods</a>. Only factors with significant differences in their concentrations (at least p<0.05) are included. Results are presented as pg or ng of cytokine per mg of total tumor protein.