Journal: PLoS ONE

Article Title: The Human Neutrophil Subsets Defined by the Presence or Absence of OLFM4 Both Transmigrate into Tissue In Vivo and Give Rise to Distinct NETs In Vitro

doi: 10.1371/journal.pone.0069575

Figure Lengend Snippet: A ) Neutrophils were immunofluorescently stained for OLFM4 (green) together with specific granule marker NGAL or azurophil granule marker CD63 (red), and DNA was labeled with DAPI (blue). Colocalization was analyzed by imaging flow cytometry and images show representative cells from the double positive populations (BF = brightfield). The diagram shows the mean colocalization score for the two fluorophores +SD from at least three experiments. The technical positive control was FITC-conjugated mouse anti-human CD16 antibody followed by Alexa Fluor 647-coupled goat anti-mouse secondary antibody and the technical negative control was FITC-conjugated mouse anti-human CD16 antibody together with DAPI, showing the minimum and maximum values that can be obtained by this analysis. The biological positive control was specific granule marker lactoferrin (LF) together with NGAL, and the biological negative control was lactoferrin together with CD63, showing the resolution of the analysis. B ) Pooled neutrophils from three donors were subjected to subcellular fractionation, and the fractions containing peak content of azurophil granule marker (MPO; fraction 1), specific granule marker (lactoferrin; fractions 10-12), gelatinase granule marker (gelatinase; fractions 13-15) and secretory vesicle marker (latent alkaline phosphatase; fraction 22) were subjected to Western blot with anti-OLFM4 antibody using rOLFM4 as a positive control. One representative blot out of three is shown. Lactoferrin (specific granule marker) and gelatinase (gelatinase granule marker) blots are also shown for fractions 10-15.

Article Snippet: CD177 was stained using a mouse anti-human CD177 antibody conjugated with phycoerythrin (Abcam), diluted 1:5, and CD16 with a mouse monoclonal anti-human CD16 antibody conjugated with fluorescein isothiocyanate (FITC) (EuroBioSciences, Friesoythe, Germany), diluted 1:20.

Techniques: Staining, Marker, Labeling, Imaging, Flow Cytometry, Positive Control, Negative Control, Fractionation, Western Blot

Journal: PLoS ONE

Article Title: The Human Neutrophil Subsets Defined by the Presence or Absence of OLFM4 Both Transmigrate into Tissue In Vivo and Give Rise to Distinct NETs In Vitro

doi: 10.1371/journal.pone.0069575

Figure Lengend Snippet: A ) Neutrophils isolated from heparinized blood were adhered to glass coverslips and either stimulated with PMA to form NETs (+PMA) or left unstimulated (-PMA). They were then left unpermeabilized (-Perm.) or permeabilized using acetone/methanol (+Perm.), after which they were immunostained for OLFM4 (red), plasma membranes (PM) were stained using FITC-conjugated WGA (green), and DNA was stained with DAPI (blue). Confocal images show representative cells or NETs from at least three individual experiments. B ) Adherent neutrophils were induced to form NETs as in A, and unpermeabilized samples were immunostained for OLFM4 (green) and MPO (red). DNA was stained with DAPI (blue). Confocal images show representative NETs from three individual experiments. A – B : Arrows indicate OLFM4-containing cells or NETs, while arrow heads indicate cells or NETs without OLFM4. The fluorophore conjugates used for each staining are indicated (AF = Alexa Fluor). The scale bars represent 5 µm.

Article Snippet: CD177 was stained using a mouse anti-human CD177 antibody conjugated with phycoerythrin (Abcam), diluted 1:5, and CD16 with a mouse monoclonal anti-human CD16 antibody conjugated with fluorescein isothiocyanate (FITC) (EuroBioSciences, Friesoythe, Germany), diluted 1:20.

Techniques: Isolation, Clinical Proteomics, Staining