[Troubleshooting] Which molecular weight FITC-Dextran is suitable for blood-testis barrier permeability experiments?

16 June 2025 1 7K Report

In the study of blood-testis barrier permeability, which molecular weight of FITC-Dextran is usually used?

When I read the literature before, I saw that the application scenarios of dextran with different molecular weights seemed to be different, such as FITC-Dextran 4kDa, 10kDa, etc. I want to confirm the more recommended model in this field~

Thank you for sharing your experience or references!

[Troubleshooting Questions are selected from MCE customer consultation emails.]

Lucian Miles

Recommended FITC-Dextran (MW 4000) [PMID: 28878076].

Protocol for FITC-Dextran (MW 4000) in Blood-Testis Barrier Permeability Assay

In Vitro SCB Model ConstructionUse Corning Biocoat human fibronectin PET inserts (3.0-μm pore size, 0.33-cm² membrane surface area) in a 24-well plate. After rehydration according to the manufacturer's protocol, seed 6×10⁴ human Sertoli cells (HSeCs) on the upper surface, and incubate with 500 μl medium in both upper and lower chambers at 37°C with 5% CO₂ and 100% humidity.

Model Integrity ValidationStart measuring transendothelial electrical resistance (TEER) using an epithelial volt/ohm meter (Evomx) and Endohm-6 chamber at 4 days after seeding, expressed as Ω/cm². Proceed with the assay when TEER readings exceed 110 Ω/cm² (typically at day 10) to confirm barrier integrity.

FITC-Dextran Permeability AssayAfter infecting the model with ZIKV or UV-inactivated ZIKV (UV-ZIKV) for 1 h, gently wash twice to remove unbound virus. Add FITC-Dextran (4-kDa, Sigma) to the upper chamber, incubate for a specified time, and collect the lower chamber supernatant. Quantify FITC-Dextran fluorescence in the lower chamber using a fluorometer, and calculate permeability as the percentage of transmigrated FITC-Dextran relative to the total input.

Control GroupsInclude mock-infected controls, UV-ZIKV-infected groups, and no-cell controls to ensure result reliability.

Notes

Cell Culture and Model SetupUse HSeCs at passages 6-9 to maintain optimal cell viability and function. Replace medium regularly during culture to sustain stable conditions.

Critical TEER MeasurementReplace medium in both chambers during each TEER measurement for accuracy. Only perform permeability assays when TEER >100 Ω/cm² to ensure barrier integrity.

FITC-Dextran HandlingAvoid light exposure during FITC-Dextran handling to prevent fluorescence quenching. Perform washing steps gently to avoid disrupting the cell monolayer.

Data Collection and AnalysisSet appropriate excitation and emission wavelengths for fluorometric detection. Repeat experiments at least three times, and present data as mean ± SEM for statistical validity.

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