Figure 1.
Differentiation of distinct M1 and M2 macrophage populations.
(A) Morphological evaluation of macrophage phenotypes. (B) Apoptosis and necrosis of macrophage populations was evaluated by staining with Annexin V and PI, respectively, followed by flow cytometry. Shown is percent necrotic/apoptotic cells (mean ± SD) from three independent experiments. (C) Expression of extracellular markers for macrophage populations as evaluated by immunostaining and flow cytometry. Relative mean flourescense intensity (MFI) ± SD of three or more independent experiments is shown, where each sample was normalized against its respective isotype control. Relevant statistically significant differences are indicated by * (p<0.05), ** (p<0.01), or *** (p<0.001), non-significant p values are indicated by n.s.
Figure 2.
The migratory ability of M1 and M2 macrophage populations.
Migration of macrophage phenotypes was evaluated by Boyden chamber experiments, where macrophages were allowed to migrate towards conditioned media from RKO or SW480 CRC cell lines, or culture medium control (RPMI+10% FBS). Mean numbers of migrating cells (n) ± SD is shown. Relevant statistically significant differences are indicated by * (p<0.05), non-significant p values are indicated by n.s.
Figure 3.
Effects on the macrophage phenotype by secreted factors from CRC cells.
(A) Morphological evaluation of macrophages stimulated with conditioned media (Cm) from RKO, SW480 or Caco2 cell lines. (B) Expression of extracellular markers was evaluated by immunostaining and flow cytometry in macrophages of M1 and M2 subtype or in macrophages stimulated with conditioned media (Cm) from CRC cell lines. Relative mean flourescense intensity (MFI) ± SD of three or more independent experiments is shown, where each sample was normalized against its respective isotype control and with the M-CSF stimulated control sample set as 1.
Figure 4.
The endocytic ability of the different macrophage populations.
Endocytosis by M1 and M2 macrophages, or macrophages stimulated with conditioned media (Cm) from RKO, SW480 or Caco2 CRC cell lines was evaluated by measuring internalization of FITC-dextran by flow cytometry. Shown is percent relative endocytosis (mean ± SD) with endocytosis by IL10 stimulated M2 macrophages set as 100%. Statistically significant differences are indicated by * (p<0.05) or ** (p<0.01). All other differences were tested but found non-significant (not indicated in figure).
Figure 5.
Macrophages are plastic cells able to switch between different phenotypes.
The morphology and expression of extracellular markers was evaluated in macrophages of distinct M1 or M2 subtypes before and after stimulation towards the opposite phenotype or stimulation by conditioned media (Cm) from RKO, SW480 or Caco2 CRC cell lines. (A) Morphological evaluation of macrophage phenotypes. (B) Expression of extracellular markers for macrophage populations as evaluated by immunostaining and flow cytometry. (C) Differentiation of M1 macrophages by stimulation with conditioned media (Cm) from CRC cell lines. Relative mean flourescense intensity (MFI) ± SD of three independent experiments is shown, where each sample was normalized against its respective isotype control and with the M1 or M2 (IL10) stimulated macrophage sample set as 100%. Relevant statistically significant differences are indicated by * (p<0.05), non-significant p values are indicated by n.s.
Figure 6.
The gene expression profiles of the different macrophage populations.
Gene expression was studied in M1 and M2 macrophages, or macrophages stimulated with conditioned media (Cm) from RKO, SW480 or Caco2 CRC cell lines using a Cytokine & Chemokine PCR Array. (A) Expression of pro-inflammatory and anti-inflammatory cytokines. (B) Expression of chemotactic factors. Shown is fold gene expression, with IL4 stimulated M2 macrophages set as 1.