results |
In vitro differentiation of myeloid-derived suppressor cells
A heterogeneous population of MDSCs were differentiated in vitro using protocols outlined by the Pullen Lab. After running flow cytometry on the cells, the results show that the cells contained either CD11b+ Ly6c+ Ly6g- or CD11b+ Ly6g+ Ly6c- which indicates the presence of a population of monocytic or granulocytic MDSCs respectively. A new cell type with phenotype CD11b+ Ly6c+ Ly6g+ was observed and is assumed to be a new type of MDSC.
MDSC differentiation of black-6 bone marrow derived stem cells from BALB/c 4T1 conditioned media
The results from flow cytometry showed that some cells present had CD11b and Ly6c/Ly6g receptors which indicates MDSCs were present in the culture. More cells with the CD11b+ Ly6c+ Ly6g+ phenotype were observed.
evaluation of IL-6 cytokine
ELISA. The IL-6 ELISA reported that small amounts of IL-6 are present in BALB/c 4T1 conditioned medium. IL-6 concentration is exponentially proportional to incubation time (see figure 3).
Because the cytokines secreted from 4T1 cells are known to affect the differentiation process and because IL-6 is present in 4T1 medium, further analysis of IL-6 was performed.
IL-6 depletion assay. A low (0.1 ml), mid (1.0 ml), and high (10 µl) concentration of IL-6 neutralizing antibody were added to Black-6 stem cells with 4T1 conditioned medium. The low concentration of IL-6 neutralizing antibody yielded more MDSCs than the mid concentration as expected. The high concentration of IL-6 neutralizing antibody did not decrease the amount of MDSCs. M-MDSCs were analyzed by graphing CD11b over Ly6C (see figure 5). G-MDSCs were also graphed using the variables CD11b and Ly6G (see figure 9).
IL-6 depletion assay. A low (0.1 ml), mid (1.0 ml), and high (10 µl) concentration of IL-6 neutralizing antibody were added to Black-6 stem cells with 4T1 conditioned medium. The low concentration of IL-6 neutralizing antibody yielded more MDSCs than the mid concentration as expected. The high concentration of IL-6 neutralizing antibody did not decrease the amount of MDSCs. M-MDSCs were analyzed by graphing CD11b over Ly6C (see figure 5). G-MDSCs were also graphed using the variables CD11b and Ly6G (see figure 9).