Evomx resistance meter

HBMEC monolayer integrity was ensured during BBB transport assays by TEER measurements and sodium-fluorescein paracellular permeability assessment, as previously described [37 (link)]. TEER readings were performed using an EndOhm^TM chamber coupled to an EVOMX resistance meter (World Precision Instruments, Inc., Sarasota, FL, USA) and sodium fluorescein levels were measured using a Hitachi F-2000 fluorescence spectrophotometer.

HUVECs were transfected with CMTM4 siRNA or adenovirus 24 h prior to seeding on a 0.1% gelatin permeable filter insert (0.4 µm pore, Falcon). Before the experiment, the resistance (R_blank) was measured by placing unseeded inserts in an Endohm-SNAP chamber filled with 5 ml of EGM-2 medium (World Precision Instruments, Berlin). The chamber was coupled to an EVOMX resistance meter (World Precision Instruments, Berlin). The transendothelial electrical resistance (TEER) was measured daily to monitor resistance buildup during growth towards full confluence. At day 2, confluent monolayers were treated with 1 U/ml thrombin for 20 min during which the TEER was measured every 5 min. After 20 min, the thrombin solution was washed away and replaced with normal medium. TEER was measured every 15 min during the restoration phase for 2 h.

For transendothelial electrical resistance (TEER) measurements 1.5 x 10⁵ cells were cultured in 12 mm diameter transwell inserts (Corning) and treated as described above. At the end of the assay, measurements were performed using an EndOhm™ chamber coupled to an EVOMX resistance meter (World Precision Instruments, Inc., USA). Results are shown in relation to average control readings, after deducting the empty insert values.