Flow cytometry and FACS

Flow cytometry and FACS on cultured erythroid cells and erythrocytes was performed as previously described.¹⁸ For assessment of ankyrin levels by flow cytometry, donor and patient erythrocytes were fixed with 1% paraformaldehyde + 0.0075% glutaraldehyde in PBS supplemented with 1 mg/mL BSA (PBSAG), 2 mg/mL glucose for 10 min at room temperature, permeabilized with 0.05% Triton X-100 for 2 min, centrifuged for 3 min at 500 G, washed once in PBSAG, re-suspended in PBSAG containing 4% BSA, and stained sequentially with BRIC272 (anti-ankyrin) and APC-conjugated polyclonal anti-mouse IgG (Biolegend), as described.¹⁸ Pronase epitope recovery experiments were performed as previously described.¹⁹ A list of antibodies used in this study is provided in the Online Supplementary Table S1. Data were acquired on a MACSQuant flow cytometer and analyzed using FlowJo v.7.6.5 (FlowJo).