Cd235a pe cy7

A three-step protocol³⁵ (link) was adapted to generate mature RBCs from mock- and LV-transduced CD34⁺ cells. From days 0 to 6, cells were grown in a basal erythroid medium supplemented with the following recombinant human cytokines: 100 ng/mL SCF (Peprotech, London, UK), 5 ng/mL IL3 (Peprotech, London, UK), and 3 IU/mL of erythropoietin (EPO) Eprex (Janssen-Cilag, Issy-Les-Moulineaux, France) and hydrocortisone (Sigma, St. Louis, MI, USA) at 10⁻⁶ M. From days 6 to 9, cells were cultured onto a layer of murine stromal MS-5 cells in basal erythroid medium supplemented only with 3 IU/mL EPO Eprex. Finally, from days 9 to 20, cells were continued to be cultured on a layer of MS-5 cells in basal erythroid medium but without cytokines. Erythroid differentiation was monitored by May Grunwald-Giemsa staining, flow cytometry analysis of the erythroid surface markers CD36 (CD36-V450, BD Horizon, Franklin Lakes, NJ, USA), CD71 (CD71-FITC, BD PharMingen, Franklin Lakes, NJ, USA) and glyophorin A (GYPA) (CD235a-PECY7, BD PharMingen, Franklin Lakes, NJ, USA). We used the nuclear dye DRAQ5 (eBioscience, San Diego, CA, USA) to evaluate the proportion of enucleated RBCs. Flow cytometry analyses were performed using the Gallios analyzer and Kaluza software (Beckman-Coulter, Brea, CA, USA).