14c sucrose

Barrier integrity or monolayer tightness was assessed by measuring transendothelial electrical resistance (TEER) while using a Millicell ERS electrode (Millipore, Bedford, MA, USA). Paracellular permeability was assessed in monolayers by measuring the diffusion profile of 0.1 µCi of [¹⁴C] sucrose (PerkinElmer, Waltham, MA, USA). Radioactivities of samples were counted using a liquid scintillation counter (PerkinElmer, Waltham, MA, USA). The permeability coefficient was calculated while using following formula [34 (link)]: $PC=\frac{dQ}{dt}\times \frac{1}{A\times C_0}$
where dQ/dt is rate of [¹⁴C] sucrose diffusion, A is area of insert, and C₀ is the initial concentration of [¹⁴C] Sucrose added in donor compartment.

Ketamine/xylazine was purchased from Sigma-Aldrich. ¹⁴C-sucrose and optiphase supermix scintillation cocktail were purchased from PerkinElmer. ³H-Sumatriptan was purchased from American Radiolabeled Chemicals Inc. Sumatriptan was generously donated by the Porreca Lab (University of Arizona; source: Abmole Bioscience). Topiramate was purchased from Cayman Chemical. TS-2 tissue solubilizer was purchased from Research Products International. EDTA-free complete protease inhibitors were purchased from Roche. The Coomassie Plus Better Bradford Assay kit was purchased from Thermo Scientific. XT sample buffer, XT reducing agent, Precision Plus dual color prestained molecular weight markers, and TGX criterion gels were purchased from Bio-Rad. Primary antibodies used for Western blotting (WB) include the following: claudin-5 [Invitrogen (Thermo Fisher), catalog #4C3C2, 1:500], occludin [Invitrogen (Thermo Fisher), catalog #OC-3F10, 1:1000], and α-tubulin (Cell Signaling, catalog #DM1A, 1:5000). Secondary antibodies for WB were purchased from Cell Signaling Technology and used at a 1:20,000 dilution. All other chemicals were purchased from Sigma-Aldrich.

Fetal Bovine Serum (FBS), Penicillin/streptomycin, glutamine, Dextran, Texas Red™ (3000, 10,000, 40,000 and 70,000 MW), B-27™ Supplement (50X), HEPES and Hoechst 33342 were from Invitrogen (Thermo Fisher, Monza, Italy). [¹⁴C]sucrose, [¹⁴C]mannitol, [³H]methyl-glucose, [³H]citalopram, [¹⁴C]leucine, [¹⁴C]phenytoin, [¹⁴C]phenylalanine, [¹⁴C]caffeine, [³H]daunomycin, [³H]testosterone, [³H] [D-Ala2]-Deltorphin II, [³H]propranolol, [³H]verapamil, [³H]prazosin and Microscint 20 Scintillation liquid were purchased from Perkin Elmer, Milan, Italy. [³H]vinblastine, [³H]L-DOPA, [³H]bupropion, [¹⁴C]taxol, [³H]gabapentin, [³H]indomethacin, [³H]raclopride, and [³H]flumazenil are from BIOTREND Chemikalien GmbH (Köln, Germany). Dimethyl sulfoxide (DMSO), lucifer yellow (LY), kynurenic acid, (GF120918), KO143, JPH203, phloretin, and triton X100 were purchased from Sigma-Aldrich, Milan, Italy. Aqueous solutions of 8% paraformaldehyde were obtained from Electron Microscopy Sciences (Hatfield, PA, USA).
Tissue-culture-treated multi-well plates and transwell filter inserts (1.12 cm² growth area, 0.4 µm pore size; transparent polyester) and Corning 96-well plates polystyrene were obtained from Corning (Sigma-Aldrich, Milan, Italy). Optiplate-96 and white Opaque 96-well microplate were from Perkin Elmer, Milan, Italy.

Fetal bovine serum, penicillin/streptomycin, glutamine, HEPES, fungizone, deoxyribonuclease I, and Hoechst 33342 were from Invitrogen (Thermo Fisher, Monza Italy). [14C]sucrose, [14C]mannitol, [3H]methyl-glucose, [14C]leucine, [14C]phenytoin, [14C]caffeine, [3H]digoxin, [3H]daunomycin, [3H]testosterone, [3H] [D-Ala2]-deltorphin II, [3H]propranolol, [3H]prazosin and Microscint 20 scintillation liquid were purchased from Perkin Elmer, Milan Italy and [3H]vinblastine, [14C]Taxol, [3H]methotrexate, [3H]gabapentin and [3H]flumazenil were from BIOTREND Chemikalien GmbH (Köln, Germany).
Fluorescein isothiocyanate-labelled FITC-dextran 40, dimethyl sulfoxide (DMSO), lucifer yellow, rhodamine-123, kynurenic acid, elacridar (GF120918), Y-27632 (ROCK inhibitor), and triton X100 were purchased from Sigma–Aldrich, Milan, Italy, and 8% paraformaldehyde aqueous solution was from Electron Microscopy Sciences (Hatfield, PA, USA).
CHDI compounds, denoted cp (A to J) and histone deacetylase (HDAC) inhibitor (cp K) were synthesized as previously described [10 (link),11 (link)].

Synthesis of [³H]-SN-38 (0.629 TBq mmol^–1) was performed by Moravek Inc (Brea, CA, USA). [¹⁴C]-sucrose (0.0215 TBq mmol⁻¹), and [³H]-diazepam (3.3 TBq mmol⁻¹) were purchased from Perkin Elmer Life sciences (Villebon, France). Elacridar (also known as GF120918), MK571, Ko143, and valspodar (also known as PSC833) were purchased from Sigma.

Following established procedures^{39 (link)}, albumin (Sigma, St. Louis MO) was labeled with ^99mTc (GE Healthcare, Piscataway, NJ). A mixture of 240 mg/ml stannous tartrate and 1 mg/ml albumin was adjusted to pH 3.0 with HCl. One millicurie of ^99mTc-NaOH₄ was added to this mixture and allowed to incubate for 20 min. The ^99mTc-albumin was purified on a column of G-10 Sephadex (GE Healthcare) in 0.1 ml fractions of phosphate buffer (0.25 M). Radioactivity in the purified ^99mTc-albumin peak was more than 90% acid precipitable in an equal volume of 1% bovine serum albumin (BSA) and trichloroacetic acid (30%). 5 × 10⁶ cpm/mouse of purified ^99mTc-albumin fraction was combined with 1 × 10⁷ dpm/mouse of ¹⁴C-sucrose (Perkin Elmer, Waltman, MA) in a final volume (0.2 ml/mouse) of lactated Ringer’s solution containing 1% BSA.