Millicell insert with 8.0 μm pores

The cell migration assay was performed using a Millicell^® insert with 8.0 μm pores (Merck Millipore, Darmstadt, Hessen, Germany) according to the manufacturer’s instructions [57 (link)]. The freshly isolated PBMCs were incubated with different concentrations of rHcES-15 (10, 20, 40, and 80 µg/mL) and similarly, the control group was treated with an equal volume of PBS and pET32a protein (10 μg/mL) for 2 h at 37 °C and 5% CO₂. The cells (200 µL) were seeded into the upper chamber and the lower chamber was filled with 1300 μL RPMI 1640 medium. Then, the cells migrated through the polycarbonate membrane into the lower chamber were determined by a Neubauer counting chamber. Each experiment was performed in triplicate.

After achieving gene knockdown, cells seeded in 12-well plates were collected and the density was adjusted to 1.5 × 10⁶ cells/mL. Migration was assayed in Millicell® insert with 8.0 μm pores (Merck-Millipore, USA) [30 (link),35 (link)]. Then 200 μL cells with the density of 1.5 × 10⁶ cells/mL were seeded in the upper chamber while the lower chamber was filled with 1300 μL 1640 cell medium. After 2 h of incubation the filters were removed and the cells that migrated through the membrane into the lower chamber were counted with a Neubauer counting chamber. The results were presented as percentages of the seeded PBMC. Each experiment was performed in triplicate.

After incubation of PBMCs with varying concentrations of rHc-TpMy (10, 20, 40 and 80 µg/mL) along with recombinant protein of pET32a and equal volume of PBS (as control) for 24 h, the migration assay was performed as described earlier [34 (link)] using a Millicell^® insert with 8.0 μm pores (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. Two hundred μL of cells (1× 10⁶ cells/mL) were seeded into the upper chamber and similarly the lower chamber was filled with 1300 μL RPMI 1640 medium and incubated for 2 h. The cells migrated through 8.0 µm pore size polycarbonate membrane into the lower chamber were determined by a Neubauer counting chamber. The results represents as mean percentage of seeded PBMCs. Each experiment was accomplished in triplicate.