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. 2018 Mar 29;9:588. doi: 10.3389/fimmu.2018.00588

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Copyright © 2018 Serwas, Huemer, Dieckmann, Mejstrikova, Garncarz, Litzman, Hoeger, Zapletal, Janda, Bennett, Kain, Kerjaschky and Boztug.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Targeted analysis of the expression of surface and granule proteins in neutrophils of specific granule deficiency (SGD) patients (A). Total protein levels of CD62L, CD11b, and CEACAM1 measured by flow cytometry in fixed and permeabilized myeloid cells in P1 (red) and P2 (orange) affected by SGD compared with two healthy controls (HCs) (black). The isotype controls for the SGD and normal cells are displayed as dashed black and gray curves, respectively. (B) CEACAM1 transcript levels in neutrophils were assessed in both patients (P1 and P2) and two HCs (HC1, shipment control; HC2, local control) and revealed a significant reduction of transcript levels as tested with one-way ANOVA (Bonferroni corrected for multiple testing, normalization to HPRT1 expression, three biological replicates shown). (C) Changes in mean fluorescence intensity (MFI) induced by fMLF stimulation (5 nM for 5 or 20 min) on the surface of live myeloid cells in P1 (red) and P2 (orange) affected by SGD compared with two HCs (black). As the monocytes and polymorphonuclear cell populations cannot be separated on the FSC/SSC channels in the SGD sample, the MFI is calculated as an average of both cell populations. (D) Elevated surface expression of CD14 and CD64 in live polymorphonuclear cells (PMNs) in SGD (right) compared with HCs (left). In the upper FSC/SSC panels, PMN (orange circle) and monocyte (green circle) populations are visible in the HCs, but these populations cannot be unmixed in the SGD sample (red circle). In the lower panel, the CD64-PE/CD14-APC stain distinguishes the monocytic CD64^high/CD14^high population (green panel) from the PMN CD64^low/CD14^low (orange panel, black circle). These populations are found back in SGD (red panel), and a PMN population is identifiable (black circle). The expression of both CD64 and CD14 is clearly higher in the PMN population in SGD versus healthy donors. (E) Immunofluorescence analysis of p22phox (green) in neutrophils of patients (P1, P2) and healthy controls (HCs) (HC1, local control; HC2, shipment control). Scale bar: 10 µm. (F,G) Analysis of p22phox (F) and gp91phox (G) by Western blot. (H) The amount of specific granule markers in a total cell lysate was compared by immunoblotting. The signal for actin serves as a loading control. The SGD and HC fractions were enriched for PMNs on a modified Ficoll gradient, and erythrocytes were removed by lysis as described. The PMN fraction contains >98% PMNs, separated on a Polymorphprep gradient.