Figure 1.
Flow cytometry gating strategy and representative morphology of reticulocyte samples.
(A) Flow cytometry dot plot showing CD71 and Hoechst staining using to define strategy gating for different reticulocyte subset sampling: CD71high, CD71medium, CD71low and CD71negative. The mean percentage of each subsets are inserted in brackets (n = 14). Cord blood reticulocyte subsets stained with new Methylene blue (see insets). (B) Morphology of cord blood reticulocytes was observed by scanning electron microscopy. The scale bars represent 1 µm. (C) The blue bordered insets show key nanostructures on the surface of the reticulocytes. The scale bars in these insets represent 100 nm. (D) Intracellular reticulocyte structures were observed by transmission electron microscopy.
Figure 2.
Immunophenotyping of reticulocyte samples.
Difference of between mean fluorescence intensity with different erythrocytic antigens and mean fluorescence intensity with secondary antibody on reticulocytes sampled from the four CD71 gates (CD71high, CD71medium, CD71low and CD71negative) (Delta of Mean Fluorescence Intensity (MFI)). The dotted red line represents the threshold of positivity based on the CD15 staining.
Figure 3.
Biomechanical properties of reticulocyte samples.
The size and biomechanical characteristics of CD71 sorted cord blood reticulocytes (different color markers represent each of the 4 isolates used). (A) The median diameter (um−/+IQR) of live reticulocytes in physiological media. (B) The median shear flow modulus (−/+IQR) (high shear flow modulus indicates reduced membrane deformability) of the reticulocyte membranes as measured by micropipette aspiration. (C) Representative micropipette aspiration images of CD71high, CD71medium, CD71low and CD71negative (top panel to bottom respectively) measured at an identical pressure. (D) The relative cytoadhesiveness of different reticulocyte samples on CHO cells (two types) and activated platelets.
Figure 4.
Metabolomic profile of reticulocyte samples.
Heat map of 14 metabolomic markers (GC-MS) found in differential amounts (dark brown indicating a greater quantity) in CD71 high, medium, low and negative reticulocyte populations. Boric acid was the only marker that increased as the reticulocyte matured from CD71 high to negative. The normalized level of marker scale is shown at the bottom of the heat map.
Figure 5.
Summary of the key phenotypic changes in maturing human cord blood samples.
An overview of the key biochemical, biophysical and metabolic changes occurring in the subsets of circulating cord blood reticulocytes relative to RNA content as measured by thiazole orange. Our finding are also put into the context of the ‘R’ reticulocyte characterization (R1 = Immature reticulocytes, R2 = Mature reticulocytes and R3 = Normocytes). [23].