Blood-Brain Barrier Mimic Permeability Assay: An In Vitro Assay to Assess the Permeability of BBB Mimic to Tracer Molecules
Published: April 30, 2023
Abstract
Source: Le Joncour, V. et al. Predicting In Vivo Payloads Delivery using a Blood-Brain Tumor-Barrier in a Dish. J. Vis. Exp. (2019)
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 …
Divulgaciones
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 |
| HuAR2T | Isolated in Dr. Dagmar Wirth Lab | https://doi.org/10.1089/ ten.tea.2009.0184 | Cultured in: EBM-2 with SupplementMix |
| Normal human primary astrocytes | Lonza | CC-2565 | Cultured in: ABM with SingleQuots |
Tags
Citar este artículo
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).