12 well culture inserts

The BBB model was established as previously reported [25 ]. bEnd.3 ​cells were plated on the upper chamber of 12-well culture inserts (0.4 ​mm pore size) (Corning, NY, USA) at a density of 1 ​× ​10⁵ ​cells per/well, and the lower chamber was filled with 1.5 ​ml of DMEM. The growth medium was changed every 2 days. For determining the integrity of BBB model, transendothelial electrical resistance (TEER; World Precision Instruments, Inc. Sarasota, FL, USA) experiment was performed. The following experiment was performed only when TEER of the monolayer reached 200 ​Ω ​cm². Additionally, J774A.1 ​cells were seed into another 12-well plates at a density of 15 ​× ​10⁴ ​cells per/well. Cell culture inserts with bEnd.3 monolayers were then transferred to 12-well plates containing J774A.1 ​cells and co-cultured for 24 ​h. SPIONs or mSPIONs were added into the cell culture inserts at a final concentration of 50 ​μg/ml. After 8 ​h of incubation, J774A.1 ​cells on the plates were washed with PBS for three times, then trypsinized, and the Fe content in J774A.1 ​cells was analyzed by ICP-MS as described in section 2.8.