This video describes a permeability assay to evaluate the permeability of in vitro blood-brain barrier using sodium fluorescein molecules.
Protocol
Measurement of the BBTB Mimic Permeability Passive diffusion of the small-molecular-weight fluorescent dye sodium-fluorescein (Na-Fl) over time from the blood to the brain side of the inserts allows the calculation of the permeability values according to the following formula: Here, dFwell is the fluorescence value measured in the well at a certain time point minus the cell culture medium …
Disclosures
The authors have nothing to disclose.
Materials
Material and Reagent
10 mL serological pipet
ThermoFisher Scientific
170361
ABM Basal Medium, 500 mL
Lonza
CC-3187
For primary human astrocytes. ABM+: contains all the additives from the supplement mix. ABM-:all the additives except for rhEGF and FBS
Basal Medium Eagle
ThermoFisher Scientific
21010046
BME-1
Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham
Gibco
21331-020
Specific to the culture of the patient-derived spheres isolated in our lab, may vary for other glioma cell lines
Greiner CELLSTAR 96 well plates S
Sigma-Aldrich
Greiner 655090
Black polystyrene wells flat bottom (with micro-clear bottom)
Fluorescein sodium salt
Sigma-Aldrich
F6377
FLUOstar Omega microplate reader
BMG Labtech
Cells
bEND3
ATCC
CRL-2299
Cultured in: DMEM (1g/L glucose) supplemented with 10% FBS, 5 mL L-glutamine and 5 mL penicillin/ streptomycin
HIFko immortalized mouse astrocytes
Isolated in Dr. Gabriele Bergers Lab
https://doi.org/10.1016/ S1535-6108(03)00194-6
Cultured in: BME-1 supplemented with 5% FBS, 5 mL 1 M HEPES, 5 mL 100 mM sodium pyruvate, 3 g D-glucose and 5 mL penicillin/ streptomycin
Blood-Brain Barrier Mimic Permeability Assay: An In Vitro Assay to Assess the Permeability of BBB Mimic to Tracer Molecules. J. Vis. Exp. (Pending Publication), e20636, doi: (2023).