| Analysis of cell phenotype and cytokine secretion by flow cytometry | Analysis of cell phenotype and cytokine secretion by flow cytometry |
| Analysis of cell phenotype and cytokine secretion by flow cytometry | Secreted cytokines and angiogenic factors were quantified in cell conditioned medium using FlowCytomiX technology (Bender MedSystems and RnD). |
| Estimation of the relative contribution of each cell population in the total cytokine production | Estimation of the relative contribution of each cell population in the total cytokine production |
| Estimation of the relative contribution of each cell population in the total cytokine production | For a given treatment, let Na, Nb and Nc be the relative number of cells present in each population (a = DN, b 2 SP, c 2 DP) and K be the amount of cytokine experimentally measured and expressed as a percentage of change of cytokine secretion to untreated cells. |
| Estimation of the relative contribution of each cell population in the total cytokine production | The coefficients were then used to infer the amount of cytokine by DN, SP and DP cell populations. |
| Patient and tissue specimens | TEM phenotype, cytokine secretion and pro-angiogenic activity were assessed by flow cytometry and in vivo or in vitro vascularization assay, respectively. |
| Reagents and antibodies | Common stocks of cytokines , inhibitors and assay reagents were used to minimize experimental variability. |
| Reagents and antibodies | Human recombinant cytokines were purchased from PeproTech (London, UK) and R&D Systems. |
| TEM from peripheral blood and tumor tissue of breast cancer patients show distinct pro-angiogenic phenotypes | Thus, CD11b+, CD14+ monocytes from patient blood and tumor tissue were referred to as “TEM” and compared with respect to receptor and cytokine expression. |
| TEM from peripheral blood and tumor tissue of breast cancer patients show distinct pro-angiogenic phenotypes | Blood and tumor TEM display a mixed M1-like (tumor-associated macrophages releasing inflammatory molecules) and M2-like (immunosuppressive macrophages polarized by anti-in-flammatory molecules) phenotype, with secretion of both the pro and antiinflammatory cytokines IL-12 and IL 10, respectively (Fig. |