Begin by preparing the differentiated endothelial cells, parasites, and astrocytes for assembling the iBBB model. After aspirating the cell culture medium from the differentiated endothelial cells, wash the cells with PBS once, then add a protease collagenase mixture to the washed cells and dissociate the cells at 37 degrees Celsius for five minutes. Transfer the protease collagenase mix with the dissociated cells into a conical tube containing hECSR, maintaining a 1:3 ratio of the mix to hECSR, then centrifuge the cells at 300 G for three minutes and aspirate the supernatant before resuspending the resultant cell pellet in the hECSR.
Dissociate the differentiated parasites with the protease collagenase mixture at 37 degrees Celsius for five minutes. Add the appropriate amount of N2B27 in a conical tube and transfer the dissociated cells in the protease collagenase mix to it, maintaining a 1:3 ratio of the mix to N2B27. Spin down the cells at 300 G for three minutes.
After aspirating the supernatant, resuspend the cell pellet in N2B27. Dissociate the differentiated astrocytes with the protease collagenase mixture at 37 degrees Celsius for five minutes. Transfer the dissociated cells in the protease collagenase mix to a conical tube containing complete astrocyte medium at a 1:3 ratio of the mix to the medium.
After centrifuging the cells at 300 G for three minutes, aspirate the supernatant and resuspend the cell pellet in complete astrocyte medium. The cells are now ready for assembling the iBBB.
