Reticulocyte stain

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)).

Packed cell volume (PCV; L/L) and total plasma protein (TPP; g/L) concentration were manually determined on the day of sampling, while automated hematological analysis (Sysmex XT-2000iV, Sysmex, Kobe, Japan) of preserved blood specimens (Streck Cell Preservative; Streck, Omaha, USA) was performed 2–8 days later (21 (link)). Automated analysis provided indicators of red blood cell number additional to PCV (red blood cell [RBC; ×10^12/L] count and hemoglobin [Hb; g/L] concentration), as well as platelet (PLT; ×10^9/L) and total white blood cell (WBC; ×10^9/L) counts. Duplicate Wrights-Giemsa-stained blood smears prepared the day of sampling were later reviewed for manual differential WBC and nucleated RBC (nRBC) counts. Reticulocyte percentage was determined from new methylene blue-stained blood smear counts (Reticulocyte stain, Sigma-Aldrich, Germany) using a Miller Square (9:1 ratio) eyepiece reticule to count the equivalent of >1,000 RBC. In addition to nRBC, the absolute reticulocyte count for each pup was subsequently calculated (reticulocyte percentage x RBC count) as a measure of the intensity of RBC regeneration.

To analyze protein expression during induced erythropoiesis, WT mice were treated with 1% PHZ (Sigma-Aldrich) in sterile PBS at day 0 (4 ml/g) and at day 3 (6 ml/g). The animals were then monitored daily, and blood was collected at different time points, as reported in the text. For all hematological studies, the blood was collected after decapitation or by tail vein puncture. During collection, blood was transferred in an EDTA-containing solution at 100 mM final concentration. The blood was analyzed in a hemocytometer (Beckman Coulter) and smeared on glass slides. For determination of reticulocytes, the blood smears were stained with reticulocyte stain (Sigma-Aldrich), based on methylene blue dye, or with May–Grünwald–Giemsa stain (Sigma-Aldrich), according to the manufacturer’s instructions. For determination of intracellular hemoglobin, the blood smears were stained with o-dianisidine (Sigma-Aldrich), as described in Fibach & Prus (2005) (link). After staining, the blood smears were air-dried and examined under a Leica stereo microscope, using a 100× oil immersion objective.