Skip to main content
. Author manuscript; available in PMC: 2019 Mar 18.
Published in final edited form as: Vet Comp Oncol. 2016 Oct 25;15(4):1309–1322. doi: 10.1111/vco.12272

Figure 5.

Figure 5.

Canine hemangiosarcoma cells produce abundant amounts of CCL2 in vitro and elicit canine monocyte migration in a CCL2-CCR2 dependent manner. Immunolabeling of DEN-HSA cells for CCL2 required pre-treatment with a protein transport inhibitor (Brefeldin A). Un-treated cells (A) displayed no immunoreactivity for CCL2, whereas cells pre-treated with Brefeldin A (B), demonstrated, strong, punctate, peri-nuclear immunoreactivity for CCL2, which is more clearly demonstrated in the enlarged single cell image inset. 40x magnification. CCL2=FITC (green). Nuclei=DAPI (blue) (C) Significant amounts of CCL2 was also detected via ELISA in conditioned media from DEN-HSA cells. Tumor cells were seeded in 24-well plates and allowed to grow for 24h prior to harvesting the supernatant for CCL2 measurement via ELISA assay. (D) Neutralization of CCL2 in HSA-conditioned media using an anti-human CCL2 antibody (5 μg/mL) resulted in a significant (~60-80%) reduction in in vitro monocyte migration as compare to the positive control of HSA-conditioned media only. Data representative of Mean ± SD. **p=0.006 unpaired one-tailed t test; ***p=0.0002, ****p<0.0001. One-way ANOVA, Tukey’s post-test.