2.9. Chemotaxis assay

The treatment of primary monocytes for each sample and the cell lines was performed in a similar way as described in the above section. The Boyden chamber (Corning, USA) system was used for this assay. After treatment, 100 µL of cell suspension in PBS with 5 × 10⁴ cells was then carefully pipetted on to the 5 µm size filter of the insert. In the 24-wells plate, the inserts were placed and each well was filled with 200 µL of PBS containing either 4 pM of Ubiquitin or CXCL12 (PeproTech, NJ, USA). The migration was allowed to take place for 2 hrs at 37°C, 5% CO₂ under either 21% or 1% oxygen in accordance with preceding experiments described above. The number of cells migrated to the lower chamber was counted using flow cytometry (BD FACS verse).