Figure 1.
Characterization of human RPMs and erythrocyte turnover. (A) Flow cytometry gating strategy used to isolate human spleen RPMs and splenic monocytes. Forward scatter and side scatter (SSC) were used to differentiate between monocyte and macrophage-containing populations of cells. From the granulocyte and macrophage-containing gate (left upper panel), the autofluorescent (AF) cells were selected (right upper panel) which were further subdivided into CD163-expressing and -nonexpressing cells (lower right panel). The CD163+AF+ cells are the RPMs. From the lower forward scatter and side scatter gate, the monocytes were isolated on the basis of CD14 and CD163 expression (lower left panel). (B) Representative cytospin micrograph of flow cytometry-sorted spleen-derived RPMs, splenic monocytes, and circulatory monocytes. (C) Z scores of genes related to iron recycling obtained by RNA sequencing (RNA-seq) on RPMs, splenic monocytes, and circulatory monocytes (n = 3). (D) Quantitative polymerase chain reaction was performed to verify RNA-seq data. Gene expression is normalized to the GAPDH housekeeping gene and expressed as relative gene expression (n = 3, mean ± SD). In line with the increased SpiC expression in RPMs, BACH1, the repressor of SpiC, was found to be downregulated. (E) Correlation between relative protein expression as determined by mass spectrometry (MS) (n = 5) and transcript abundance as determined by RNA-seq (n = 3) by RPMs. Several genes of interest are shown in red. (F) Quantification of the percentage of RPMs with ≥1 erythrocyte inclusions as depicted in Figure 1B (n = 3-6). (G) Representative benzidine staining of human RPMs depicting relatively intact adhering and internalized erythrocytes. (H) Ghost erythrocytes were detected by F-actin staining (phalloidin) in combination with antiglycophorin-A staining for the erythroid-specific marker (antiCD235a). (I-J) Conventional light microscope micrograph and benzidine staining depicting erythrocytes and erythrocyte ghosts (red arrows). (K-L) Flow cytometric gating strategy used to quantify ghost erythrocytes in the circulation of healthy individuals and in human spleen tissue. White lines mark border cropping of the image because the output file was too large. *P < .05; **P < .01 (mean ± SD). Circ, circulating; macro, macrophage; mono, monocyte; ns, not significant; Spl., spleen.

Characterization of human RPMs and erythrocyte turnover. (A) Flow cytometry gating strategy used to isolate human spleen RPMs and splenic monocytes. Forward scatter and side scatter (SSC) were used to differentiate between monocyte and macrophage-containing populations of cells. From the granulocyte and macrophage-containing gate (left upper panel), the autofluorescent (AF) cells were selected (right upper panel) which were further subdivided into CD163-expressing and -nonexpressing cells (lower right panel). The CD163+AF+ cells are the RPMs. From the lower forward scatter and side scatter gate, the monocytes were isolated on the basis of CD14 and CD163 expression (lower left panel). (B) Representative cytospin micrograph of flow cytometry-sorted spleen-derived RPMs, splenic monocytes, and circulatory monocytes. (C) Z scores of genes related to iron recycling obtained by RNA sequencing (RNA-seq) on RPMs, splenic monocytes, and circulatory monocytes (n = 3). (D) Quantitative polymerase chain reaction was performed to verify RNA-seq data. Gene expression is normalized to the GAPDH housekeeping gene and expressed as relative gene expression (n = 3, mean ± SD). In line with the increased SpiC expression in RPMs, BACH1, the repressor of SpiC, was found to be downregulated. (E) Correlation between relative protein expression as determined by mass spectrometry (MS) (n = 5) and transcript abundance as determined by RNA-seq (n = 3) by RPMs. Several genes of interest are shown in red. (F) Quantification of the percentage of RPMs with ≥1 erythrocyte inclusions as depicted in Figure 1B (n = 3-6). (G) Representative benzidine staining of human RPMs depicting relatively intact adhering and internalized erythrocytes. (H) Ghost erythrocytes were detected by F-actin staining (phalloidin) in combination with antiglycophorin-A staining for the erythroid-specific marker (antiCD235a). (I-J) Conventional light microscope micrograph and benzidine staining depicting erythrocytes and erythrocyte ghosts (red arrows). (K-L) Flow cytometric gating strategy used to quantify ghost erythrocytes in the circulation of healthy individuals and in human spleen tissue. White lines mark border cropping of the image because the output file was too large. *P < .05; **P < .01 (mean ± SD). Circ, circulating; macro, macrophage; mono, monocyte; ns, not significant; Spl., spleen.

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