70 kd fluorescent dextran

A single cell-derived clone of HeLa cells (ATCC, Manassas, VA; mycoplasma negative and validated by STR profiling) were maintained in OptiMEM (Gibco/ThermoFisher) with 4% FBS and plated on uncoated optical glass-bottom 96 well plates, at appropriate densities to reach 70–90% confluence on the day of the transport assay. To permeabilize, cells were rinsed for 2 min in ice-cold PBS, and permeabilized on ice for 10 min in 15–30 µg/mL digitonin (Calbiochem, San Diego, CA) in permeabilization buffer (PRB, 20 mM HEPES, 110 mM KOAc, 5 mM Mg(OAc)2, 0.5 mM EGTA, 250 mM sucrose, pH 7.5, with protease inhibitor cocktail). Following permeabilization, cells were placed on ice and rinsed 3 × 5 min in transport buffer (TRB, 20 mM HEPES, 110 mM KOAc, 2 mM Mg(OAc)₂, 5 mM NaOAc, 0.5 mM EGTA, 250 mM sucrose, pH 7.3, with protease inhibitor cocktail). The optimal digitonin concentration varied by cell density and passage number, and was optimized prior to each set of assays for the ability to permeabilize the majority of plasma membranes while maintaining nuclear exclusion of a 70 kD fluorescent dextran (ThermoFisher).

All animal procedures were approved by the Johns Hopkins Animal Care and Use Committee. Timed pregnant C57BL/6J females (Jackson Laboratory, Bar Harbor, ME) were sacrificed by cervical dislocation at E16, cortex dissociated, and cells plated at 50,000/well on poly-D-lysine/laminin-coated, optical glass-bottom 96-well plates. Growth medium consisted of Neurobasal supplemented with B27, Glutamax, and penicillin/streptomycin (Gibco/ThermoFisher). At 5–7 days in vitro, neurons were rinsed in prewarmed PBS and permeabilized for 4 min. at 37° in a hypotonic solution containing 0–40 µM Tris-HCl pH 7.5 (to cause osmotic swelling) and 50–150 mg/ml BSA (for molecular crowding/mechanical support). Following permeabilization, cells were placed on ice and rinsed 2 × 5 min in transport buffer (TRB, 20 mM HEPES, 110 mM KOAc, 2 mM Mg(OAc)₂, 5 mM NaOAc, 0.5 mM EGTA, 250 mM sucrose, pH 7.3, with protease inhibitor cocktail). All rinse and assay buffers were supplemented with 50 mg/mL BSA. The optimal hypotonic buffer and BSA concentration varied by batch, and was optimized prior to each set of assays for ability to permeabilize the majority of plasma membranes while maintaining nuclear exclusion of a 70 kD fluorescent dextran (ThermoFisher).