American Society of Hematology

Figure 1.

$Figure 1. Peripheral blood cell purification flowchart. Reticulocytes and erythrocytes were purified from the peripheral blood of healthy donors. Blood was centrifuged for 10 minutes at 150g to remove plasma and a portion of the leukocytes and platelets. During this step, crude cell populations were sampled for MS analysis. The remaining leukocytes and platelets were removed by passing the cells through a cellulose column packed in 2-mL plastic syringes. Cells not retained in the column were separated into 2 fractions that were centrifuged through Percoll layers of different concentrations to obtain fractions depleted (P3) or enriched (P4) in reticulocytes. A cell sample from P3 (EEP) was used for MS analysis. In the experiment reported, the EEP cell population contained 0.62% of reticulocytes. Cells were labeled with thiazole orange (TO), and erythrocytes (EPP; TO− cells) and reticulocytes (Retic; TO+) were purified by FACS from P3 and P4 fractions, respectively, using a FACSJazz cell sorter (BD Biosciences). Between 750 000 and 2 million purified reticulocytes and between 6 and 15 million erythrocytes were obtained from each purification procedure and used for subsequent analyses. The purity of isolated cell populations was controlled by cytocentrifuged cells stained with New Methylene Blue (reticulocyte stain; Sigma-Aldrich).$

Peripheral blood cell purification flowchart. Reticulocytes and erythrocytes were purified from the peripheral blood of healthy donors. Blood was centrifuged for 10 minutes at 150g to remove plasma and a portion of the leukocytes and platelets. During this step, crude cell populations were sampled for MS analysis. The remaining leukocytes and platelets were removed by passing the cells through a cellulose column packed in 2-mL plastic syringes. Cells not retained in the column were separated into 2 fractions that were centrifuged through Percoll layers of different concentrations to obtain fractions depleted (P3) or enriched (P4) in reticulocytes. A cell sample from P3 (EEP) was used for MS analysis. In the experiment reported, the EEP cell population contained 0.62% of reticulocytes. Cells were labeled with thiazole orange (TO), and erythrocytes (EPP; TO⁻ cells) and reticulocytes (Retic; TO⁺) were purified by FACS from P3 and P4 fractions, respectively, using a FACSJazz cell sorter (BD Biosciences). Between 750 000 and 2 million purified reticulocytes and between 6 and 15 million erythrocytes were obtained from each purification procedure and used for subsequent analyses. The purity of isolated cell populations was controlled by cytocentrifuged cells stained with New Methylene Blue (reticulocyte stain; Sigma-Aldrich).

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