Purification of Reticulocytes from Blood

Reticulocytes were purified from umbilical cord blood or hemochromatosis blood samples within 48 hours of collection, following previously described protocols (Borlon et al., 2012 (link)). In brief, a blood sample aliquot was stored for future DNA extraction and genotyping. In the case of hemochromatosis blood, samples were typed for ABO blood type and Duffy phenotype (Fy) using standard serological methods (ABO/Rh Blood Typing Kit, Edulab and DiaMed-ID Micro Typing Systems, DiaMed, respectively). Leukocytes were removed using filters (Fresenius Kabi) and the reticulocytes were then concentrated by centrifugation (15 minutes at 2800 rpm, without a brake) through a Percoll gradient. The optimal Percoll gradient was selected for each sample after testing gradients ranging from 66%-74% on a small volume. The proportion of purified reticulocytes was calculated by light microscopy from a thin smear with new methylene blue staining (Reticulocyte stain, Sigma). Cells with two or more granules of reticulin were considered to be reticulocytes. Reticulocyte purity was in a range of 15%-90%, and only samples with a proportion of reticulocytes >25% were used in invasion assays. Freezing and thawing of reRBC samples was performed as previously described (Borlon et al., 2012 (link)).