Setup of the μ-slide cell co-culture chemotaxis assay. (A) semi-quantitative PCR analysis of mock-CHO and ACKR3-CHO cells. (B) Microphotographs of EGFP-positive ACKR3-CHO cells. (C) Schematic representation of the μ-slide chamber. HUVECs (hereafter referred to as ECs) adherent to the central observation area are exposed to three different experimental conditions: no CXCL12 (–/–), addition of 50 ng/ml of CXCL12 to one (+/–) or both (+/+) lateral reservoirs. The horizontal arrow indicates the anticipated direction of HUVEC migration in the (+/–) experimental condition. (D) Quantification of forward migration index (FMI) and displacement of centre of mass (COM) parameters measured for HUVECs treated as illustrated in (C). (E) Schematic representation of the μ-slide chamber in the presence of mock-CHO cells. HUVECs were seeded in the central observation area whereas mock-CHO cells were seeded in both lateral reservoirs. After 4 h, cells were left untreated (–/–) or were incubated with 50 ng/ml CXCL12 added to one (+/–) or both (+/+) lateral reservoirs. (F) At the end of the incubation, FMI and COM parameters were calculated. (G) Schematic representation of the μ-slide chamber in the presence of ACKR3-CHO cells. HUVECs were seeded in the central observation area whereas ACKR3-CHO cells were seeded in both lateral reservoirs. After 4 h, cells were left untreated (–/–) or were incubated with 50 ng/ml CXCL12 added to one (+/–) or both (+/+) lateral reservoirs. At the end of the incubation, FMI and COM parameters were calculated (H). Data in panels (D,F,H) are the mean ± S.E.M of two independent experiments. *p < 0.05 or better, Student t-test.
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