Release of PS-exposed, inside-out autophagic vesicles is a normal mechanism of reticulocyte maturation. (A) Trypsin-treated in vitro produced reticulocytes were fixed and stained with an anti–trypsin-insensitive GPA antibody (red, BRIC 256) before permeabilization, and then an anti–trypsin-sensitive GPA antibody after permeabilization (green, R10), shown with higher magnification and 3D reconstruction using Volocity software (Perkin Elmer, Waltham, MA). (B) Trypsin-treated in vitro-produced reticulocytes were fixed and stained for trypsin-insensitive GPA (green, R10) and autophagic marker LC3 (red). (C) Trypsin-treated red cells from peripheral blood stained as in (A). Shown in merge and magnification of a maximum projection. (D) Live imaging of in vitro-produced reticulocytes stained for PS, intracellular GPA (BRIC163), and intracellular AE1 (BRIC155) (all green). (E) Fixed and permeabilized in vitro-produced reticulocytes stained for PS (green) and giantin (red). Staining is shown in fluorescence and phase overlay. (F) Fixed unpermeabilized in vitro-produced reticulocytes stained for extracellular AE1 (red, BRAC18) and intracellular GPA (green, BRIC163) followed by Mitotracker (blue) staining post-permeabilization. Shown is a composite of a 2D slice in phase, fluorescence, and overlay images, and 3D reconstructions of the vesicle using Volocity software. All scale bars are 5 µm.