Migration Assay for Recombinant Protein

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.