Abstract
Human neutrophils are filled with intracellular storage organelles, called granules and secretory vesicles, which differ in their content of soluble matrix proteins and membrane-bound molecules. To date, at least four distinct granule/vesicle subsets have been identified. These organelles may secrete their content extracellularly following mobilization to and fusion with the plasma membrane, but some of them may also fuse with internal membrane-enclosed organelles, typically a plasma membrane-derived phagosome. There are also instances where different granules appear to fuse with one another, a process that would enable mixing of their matrix and membrane components. Such granule fusion enables e.g., myeloperoxidase-processing of intragranular oxygen radicals, a key event in the formation of neutrophil extracellular traps (Björnsdottir et al., 2015) [1]. Described herein are data that show the quantification of such heterotypic granule–granule fusion by the use of imaging flow cytometry, a technique that combines flow cytometry with microscopy. The analysis described is based on immunofluorescent staining of established granule markers (lactoferrin and/or NGAL for one granule subset; the specific granules, and CD63 for another granule subset, the azurophil granules) and calculation of a colocalization score for resting and PMA-stimulated neutrophils.
Highlights
Experimental setupHuman granulocytes were isolated from buffy coats as described by Bøyum [20], and diluted to 1 Â 107 cells/ml in Krebs-Ringer phosphate buffer (KRG) containing glucose (10 mM), Mg2 þ (1.5 mM) and Ca2 þ (1 mM)
Human neutrophils are filled with intracellular storage organelles, called granules and secretory vesicles, which differ in their content of soluble matrix proteins and membrane-bound molecules
Biology Phagocyte biology Imaging flow cytometry data, image analyses Imaging flow cytometry; ImageStreamX MkII Analyzed data, raw data (.cif and .daf files from one representative experiment) Isolated human neutrophils were kept on ice or treated with phorbol myristate acetate (PMA) for 3 min at 37 °C
Summary
Human granulocytes were isolated from buffy coats as described by Bøyum [20], and diluted to 1 Â 107 cells/ml in Krebs-Ringer phosphate buffer (KRG) containing glucose (10 mM), Mg2 þ (1.5 mM) and Ca2 þ (1 mM). Indirect immunofluorescent staining of cells was performed essentially as described in [21]. Cells were fixed and permeabilized with a fixation/permeabilization kit (BD) and incubated with combinations of the following primary antibodies; lactoferrin (DAKO, 89 μg/ml), CD63 (Sanquin, 2 μg/ ml), and/or NGAL (Abcam, 5 μg/ml). Samples were incubated with secondary antibodies (F(ab') fragments; 4 μg/ml) conjugated with Alexa Fluor 488 or 647 in combinations as is indicated in the figures. Samples were diluted in 20 μl PBS and supplemented with DAPI (3 μM) for staining of DNA. The raw data from one representative experiment can be found as Supplemental data
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