Fluorescein ex protein labeling kit

Abx was labeled with FITC using fluorescein-EX protein labeling kit (Thermo Fisher). A 0.5 mL solution of 2 mg/mL abraxane was mixed with 50 µl of 1 M bicarbonate and incubated with the reactive dye for 1 h at room temperature. The labeled Abx was purified from free dye using a spin column (MW > 20 kDa). Cells (1 × 10⁵ cells/well of a four-well chamber) were incubated with 1% bovine serum albumin at 24°C for 30 min and then with 5 μg/mL of FITC-labeled Abx at 37 °C for 1 h. Cells were fixed with 4% paraformaldehyde followed by nuclear staining with 4', 6-diamidino-2-phenylindole (DAPI) after washing and observed under a confocal microscope (Nanoscope, Daejeon, Korea), or cells were analyzed using Attune NxT flow cytometer (Thermo Fisher). For the internalization inhibition, cells were incubated with 50 μM of 5-(n-ethyl-n-isopropyl) amiloride (EIPA, Thermo Fisher), 50 μM of chlorpromazine (CPZ, Sigma-Aldrich), and 100 μM of anti-IL4Rα blocking antibody or anti-IgG antibody (R&D Systems, Minneapolis, MN) for 30 min prior to incubation with FITC-labeled Abx.

The ex vivo permeation studies were carried out in a Franz Cell system (Hanson Research, Chatsworth, LA, United States) with a diffusion area of 1 cm². The Franz Cell system was maintained at a constant temperature of 37°C by thermostatic bath circulation, while the receptor medium (0.9% NaCl) was stirred constantly at 350 rpm during the experiments. For the permeation studies, pig skin was purchased at Minimally Invasive Surgery Center Jesús Usón (Cáceres, Spain). Explants of 1 cm² were prepared and carefully placed at the interface between the donor and receptor compartments. Aliquots (100 μl) of LysRODIΔAmi (15.84 μM), previously labeled with fluorescein using the kit Fluorescein-EX Protein Labeling Kit (Thermo Fisher Scientific, Madrid, Spain), were placed over the skin explants and samples were collected after 6 h. The skin was removed and the layers were separated with a surgical scalpel. The protein was extracted from the different layers and subsequently analyzed by fluorescence spectroscopy (excitation/emission 494/518 nm).