Chemotactic chamber

Chemotaxis was analysed as previously described [21 (link)]. Briefly, freshly isolated human monocytes or cryopreserved monocytes were resuspended in medium at a concentration of 0.5 × 10⁶ cells/ml. Subsequently, monocytes were placed to the upper wells of a chemotactic chamber (Neuroprobe) and migrated towards chemotactic growth factors which were added to the lower wells of the chamber. Upper and lower wells were separated by a polycarbonate membrane (pore size 5 μm, Millipore). The cells were allowed to migrate for 90 min in a humidified incubator (5 % CO₂) at 37 °C. Adherent cells on polycarbonate membrane were fixed for 10 min using absolute ethanol and stained with Giemsa dye. The non-migrated cells from the upper side of the membrane were scraped off gently with a cotton bud. Migrated cells were quantified by counting cells in five high-power fields (20× primary magnification) of four different wells per condition.

EGF (Millipore Chemicon, United States) diluted with binding medium (DMEM, 0.1%BSA, 25 mM HEPES) at the concentration of 0 ng/ml, 1 ng/ml, 10 ng/ml, and 100 ng/ml were placed in the lower chamber of the chemotactic chamber (Neuro Probe, China), with binding medium added as the negative control. About 5 ×10⁵/ml MDA-MB- 231 cells were re-suspended with DMEM medium and added into the upper chamber of the chemotactic chamber. Polycarbonate films (Neuro Probe, China) treated overnight with Fibronectin (Sigma, United States) at 4°C were placed between the upper and lower chambers of chemotactic cells. Then the chemotactic chamber was incubated for 3 h in a 37°C incubator with 5% CO₂. The cells passing through the membrane were fixed and stained, subsequently counted in three fields randomly by microscope.