Isolation of Loa loa Microfilariae

Whole blood samples (4 ml) were collected from a donor infected with L. loa and MF were obtained using version of a previously described protocol [20 (link), 21 (link)]. In brief, 2 ml of whole blood was layered onto 2 ml-modified Percoll gradient (Sigma-Aldrich) in a 15-ml tube (Falcon) and centrifuged at 2000× rpm for 20 min without brake using a bench-top centrifuge (Human Diagnostics, Wiesbaden, Germany). Using a Whatman^® filter paper (pore size 5 µm) (Merck Millipore, Tullagreen, Ireland) placed in a filter paper holder, a dropper was used to discard the topmost part containing the serum. Then, the filter was mounted on another 15-ml tube and the whitish area containing the parasite was then filtered using a syringe (Terumo, Tokyo, Japan). The filter paper was then removed with a sterile pipette and placed in a Petri dish (Falcon) containing RPMI medium (Sigma-Aldrich) to aid migration of MF out of the paper into the medium. MF number and motility was determined using a dissecting microscope Leica M80 (Leica, Singapore, Republic of Singapore). MF were either frozen at − 80 °C for antigen preparation or used for infection of wildtype BALB/c mice.