Migration assay

One of the most commonly used migration assays is the Boyden chamber/Transwell assay (Figure 2.2). This method is used to quantify the migration of cells exposed to chemoattractants such as chemokines. The chamber includes two compartments separated by a microporous filter (8 µm) through which the cells migrate.

The relevant chemoattractant solution is placed in the lower chamber to create a chemotactic gradient while the cells to be tested are incubated in the upper chamber. After an appropriate incubation time, the upper surface of the filter is scraped twice with cotton swabs to remove non-migrating cells. Migrated cells on the lower surface of the membrane are fixed and stained by Diff-Quick^® kit. Five to seven random photographs were taken of each membrane with an Olympus light-microscope coupled with a camera, and the cells in each photograph were counted and quantified using the “ImageJ” software.

Figure 2.2: A ThinCertTMcell culture insert is placed in the well of a multiwell cell culture plate, thus forming a migration chamber. The migration chamber consists of an upper and lower compartment with a porous PET membrane in -between. Cells may actively migrate from the upper to the lower compartment. The figure was taken and modified from (Oppegard et al. 2010).

51

2.3.7.1 Determination of optimal concentrations of CXCL12 and AMD3100 Best concentration of CXCL12

This experiment was performed with the CXCR4-positive tumour cell line (ZMK-1). 8 x 10⁴ cells were seeded into the insert. CXCL12 as chemoattractant was added to the lower chamber in the concentrations 0, 25, 50 and 100 ng/ml. The cells were allowed to migrate for 26 hours at 37°C and 5% CO2.

Best concentration of AMD3100

This experiment was also performed with ZMK-1 cell line. The cells were incubated with six different concentrations of AMD3100, i.e. 0, 1, 5, 12.5, 25 and 50 µg/ml for 26 hours at 37°C and 5% CO2. At the end of the incubation time 8 x 10⁴ cells were seeded into the insert.

CXCL12 (100 ng/ml, optimal concentration determined in the experiment described above) was added to the lower chambers. The cells were allowed to migrate for 26 hours at 37°C and 5% CO2.

2.3.7.2 Influence of irradiation and CXCL12 on cell migration

Cells were initially starved of serum for two hours. A cell suspension containing 16 x 10⁴ cells was prepared and kept in four 15 ml plastic tubes. One tube contained cells for the control group (non-irradiated cells); the second tube was irradiated at 0.5 Gy; the third and fourth tubes were irradiated at 2 and 4 Gy, respectively. 500 µl cell suspensions aliquots from each tube (8 x 10⁴ cells) were seeded into serum-free medium in the two inserts. To investigate the influence of CXCL12 on cell migration, 100 ng/ml of CXCL12 was added to the one of the lower chambers (see Table 2.13, A1-A4, purple area). The remaining lower chambers were CXCL12-free (Table 2.13, B1-B4, green area).

The cells were allowed to migrate for 26 hours at 37°C and 5% CO2. The experiments were repeated in duplicate for all cell lines. The results are expressed as the mean number of migrated cells ± standard deviation. Statistical differences were determined by Student’s t-test. A value of p<0.05 was considered to indicate a statistically significant difference.

52

2.3.7.3 Influence of AMD3100 on CXCR4-positive migrating cells

This experiment was only performed with the CXCR4-positive tumour cell lines (ZMK-1 and FaDu). Cells were initially starved of serum for two hours. A cell suspension containing 16 x 10⁴ cells was prepared and maintained in eight 15 ml plastic tubes. Two tubes contained cells for the non-irradiated control group. For each radiation dose (0.5, 2 and 4 Gy) two tubes were prepared, i.e. for each dose one tube was treated with AMD3100 (5 µg/ml) for 30 minutes at 37°C and 5% CO₂ and the other tube was untreated (see Table 2.14, purple area: treated and green area: untreated cells). To control for AMD3100 influence on non-irradiated cells one of the control tubes was also treated with AMD100 (see Table 2.14, blue area).

1 2 3 4 5 6

Table 2.13: Schematic illustration of a 24 -well transwell plate seeded with irradiated and non -irradiated cells.

100 ng/ml of CXCL12 was added in the lower chamber of purple area, which was not available in green area.

Table 2.14: Schematic illustration of a 24 -well transwell plate seeded with irradiated and n on-irradiated cells. 100 ng/ml of CXCL12 was added in all lower chambers except A1 for control. Irradiated cells in the purple area were treated with 5µg/ml of AMD3100. However, irradiated cells in the green area were untreated.

53

After the six treated cell suspensions had been irradiated, 500 µl cell suspension (8 x 10⁴ cells) aliquots from all nine tubes were seeded into serum-free medium. To investigate the inhibitory effect of AMD3100 on cell migration, 100 ng/ml of CXCL12 was added to all lower chambers except one control chamber (see Table 2.14, A1). The cells were allowed to migrate for 26 hours at 37°C and 5% CO2. The experiments were repeated in triplicate.

2.3.7.4 Data analysis

The measurements and numbers of migrated cells were determined in duplicate to analyse the impact of the CXCL12 gradient on cell migration. The experiments on the inhibitory effect of AMD3100 on CXCR4-positive cell lines were repeated three times. The results are expressed as the mean number of migrated cells ± standard deviation. The differences between two groups were compared by Student´s t-test. One-way ANOVA was used to compare three or more groups. A value of p<0.05 was considered to indicate a statistically significant difference.

54