Cy5 conjugated goat anti rabbit igg h l f ab 2 fragments

CD45^– bone marrow erythroid cells (3 × 10⁵ cells) were fixed with 4% paraformaldehyde and blocked with 1% BSA-C solution (Aurion) for 1 h. Subsequently, samples were permeabilized with 0.3% Triton X-100 in phosphate buffered saline and incubated with a rabbit anti-LC3 polyclonal antibody (Novus Biologicals, Littleton, CO), then, subsequently a Cy5-conjugated goat anti-rabbit IgG (H+L) F(ab’)₂ fragments (Invitrogen, Carlsbad, CA). After incubation and wash, samples were stained with FITC conjugated rat anti-LAMP-1 monoclonal antibody (Biolegend, San Diego, CA). Fluorescent signal was observed using an Olympus FluoView 1000 confocal microscope (Olympus, Tokyo, Japan). Illustration was captured using a 60 × objective lens for 10 field per sample by sequential field collection and discard 10 fields. A total of 50 cells per sample were analysed. Image analysis and calculation of Pearson's correlation coefficients and confidence intervals were undertaken as previously described^{17 (link),42 (link)}. Autophagosomes with punctate LC3 expression in erythroblasts were count and calculated as the percentage of autophagic cells in total erythroid cells (Supplementary Fig. S4).

CD45^– human bone marrow erythroblasts were isolated and analysed co-localization of LC3/LAMP-1 using confocal microscope^{17 (link),42 (link)}. CD45^– human bone marrow erythroblasts (3 × 10^5t5 cells) were fixed and stained with a rabbit anti-LC3 polyclonal antibody (Novus Biologicals), then, subsequently a Cy5-conjugated goat anti-rabbit IgG (H+L) F(ab’)₂ fragments (Invitrogen) as secondary antibody, after incubation and wash, samples were incubated with a FITC conjugated rat anti-LAMP-1 monoclonal antibody (Biolegend). After incubation and wash, samples were stained with 4′,6-Diamidino-2-phenylindole, dihydrochloride (DAPI, Molecular Probes, Eugene, OR). Images were captured using a 60 × objective lens for ≥ 10 field per sample. A total of 50 erythroid cells per sample were analysed. Fluorescent signal was observed and analysed as described in the section of “Confocal microscopic analysis of murine erythroid autophagy” (Supplementary Figs. S8 and S9).