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. 2014 Jun 17;5(14):5350–5368. doi: 10.18632/oncotarget.2110

Fig.2. Hypoxia Primed Breast Cancer Cells Release Soluble Mediators to Chemoattract Macrophages and Migrated Macrophages Exhibit M2-polarized phenotype.

Fig.2

(A) THP-1 derived macrophages previously cultured on PET cell culture inserts were introduced in upper well of modified boyden chamber while lower well contained conditioned media from hypoxia primed (3 and 6hrs) breast cancer cells (MDA-MB-231 and MCF-7). Extent of macrophage migration was evaluated after maintaining the cultures for further 24 hrs under standard cell culture conditions. Photomicrographs depicts directional migration of macrophages towards hypoxic breast cancer cells conditioned media as evaluated through Geimsa staining of migrated macrophages followed by bright field microscopy (100X). The Quantification of macrophage migration was done by DAPI staining of migrated macrophages followed by counting of nuclei in five different fields of three replica wells. (B) Representative photomicrographs depicting M2-polarization of migrated macrophages as measured through immunocytochemical detection of M2 specific markers viz. CD163 and CD206. Data presented as Mean±SEM; n=5; Symbols indicate statistical significance at p < 0.05 (*).