CellXVivo Human M1 Macrophage Differentiation Kit

Optimized Base Media in CellXVivo™ Macrophage Differentiation Kits Results in More Efficient Differentiation of M1 and M2 Macrophages. The Serum-free Base Media included in the CellXVivo^™M1 and M2 Macrophage Differentiation Kits (Catalog # CDK012 and Catalog # CDK013) was screened against other commercially available base media (Competitor 1, Competitor 2) for its ability to promote M1 and M2 differentiation. Human peripheral blood CD14⁺monocytes were cultured in either Serum-free Base Media from the CellXVivo^™Macrophage Differentiation Kits, Competitor Media 1, or Competitor Media 2. M1 and M2 macrophage differentiation was performed in each of the base media with cytokines from the CellXVivo^™M1 and M2 Macrophage Kits added to each media condition in parallel. Following the differentiation protocol, M1 and M2 macrophages from each media condition were stimulated with 1 µg/mL LPS for 24 hours. Cell culture supernatants were assessed for IL-10 and IL-12 secretion using Human IL-12 p70 and Human IL-10 Quantikine^®ELISA Kits (Catalog # HS120and Catalog # D100B, respectively). These data indicate that monocytes differentiated using the Serum-free Base Media included in the CellXVivo^™Macrophage Differentiation Kits produce more efficient and pure populations of the desired M1 or M2 phenotype. Competitor base media resulted in either a mixed M1/M2 population (Competitor 1) or lower levels of cytokine secretion (Competitor 2).

Phenotypic Analysis of Human M1 Macrophages.  Flow cytometry data show cell surface marker expression of human peripheral blood CD14⁺monocytes following differentiation using reagents included in the CellXVivo^™Human M1 Macrophage Differentiation Kit. On day 6 of the differentiation, cells were harvested and stained with antibodies for CD14, CD80, CD163, and CD206 (open histograms). Cell staining was gated using isotype control antibodies (filled histograms). M1 macrophages display a CD14⁺CD80⁺CD163^dimCD206⁺phenotype.

Differentiated Human M1 Macrophages Secrete IL-12.  Human peripheral blood CD14⁺monocytes were differentiated for 6 days under M1 or M2 macrophage polarization conditions using reagents included in the CellXVivo^™Human M1 Differentiation Kit or the CellXVivo^™M2 Macrophage Differentiation Kit (R&D Systems, Catalog # CDK013). On day 6, M1 and M2 macrophages were stimulated with 1 µg/mL LPS for 24 hours. Cell culture supernatant was collected and cytokine secretion was determined using the Human IL-12 p70 Quantikine^®HS ELISA Kit (Catalog # HS120) and the Human IL-10 Quantikine^®ELISA Kit (Catalog # D100B).