Analysis of cytokine expression in TC-1 tumors. RNA samples from CD45+ and CD45-sorted cells were used for cytokine expression analysis. A. Qualitative cytokine mRNA expression analysis in total RNA samples from tumors 20 days post-injection. HPRT (Hypoxanthine Guanine Phosphoribosyltransferase) was used as control. C+ -positive control RNAs from cells stimulated with different cytokines. No -reactions without addition of cDNA. B. Quantitative Real-time PCR analysis of IL-10 expression in CD45+ and CD45- tumor fractions compared to RNA from total tumor population. Relative expression ratio is calculated based on ΔΔCt method. C. Results from SuperArray RT-PCR. Data is presented as the ratio of the housekeeping genes Ct (HKG) and the target gene Ct (TG). Ratios above 0.6 were defined as positive expression (dashed line). Light gray bars - CD45+ cells cDNA, and dark gray bars - CD45- cells cDNA. Asterisks indicate p < 0.05 tested by t-test comparing CD45+ and CD45- expression. D. IL-10 intracellular staining. Cell suspensions were stained with anti-CD11b and anti-F4/80 before fixation and with anti-IL-10 after fixation and permeabilization. FACS analysis was performed in a FACSCalibur, where the CD11b+ population (top histogram, R1) was used to define the R2 region in the FSCxSSC plot. The CD11b × F4/80 plot was gated on R2 and the histograms representing IL-10 expression were gated on R1 and R2 and R4 or R5, CD11b+F4/80+ macrophages or the CD11b+ population, respectively; IL-10 expression in the tumor cells corresponds to cells gated on R7. Result representative of 3 independent experiments.
Bolpetti et al. BMC Immunology 2010 11:27 doi:10.1186/1471-2172-11-27