Fig. 1

mmp9:Citrine identifies mature neutrophils.

a Confocal images of triple transgenic larvae Tg(lysC:CFP-NTR)^vi002/ Tg(BACmmp9:Citrine-CAAX)^vi003/ Tg(mpeg:mCherry)^gl23 reveal myeloid cells with different expression levels of the three analyzed markers. Stitched whole-mount images of a 5 dpf (days post fertilization) larva. n = 3. b High magnification showing presence of three different cell types: #1 neutrophil: lysC:CFP⁺/ mpeg:mCherry⁺/  mmp9:Citrine⁺; #2 neutrophil: lysC:CFP⁺/ mpeg:mCherry^-/ mmp9:Citrine^-; #3 macrophage: lysC:CFP^-/ mpeg:mCherry⁺/ mmp9:Citrine^-. c Flow cytometric analysis (BD LSRFortessa) of myeloid cells from mpeg:mCherry^-/ lysC:CFP⁺ control larvae, lysC:CFP^-/ mpeg:mCherry⁺ controls, or triple transgenic larvae lysC:CFP⁺/ mmp9:Citrine⁺/ mpeg:mCherry⁺(dot plots left to right) at 3 dpf. Histogram and box plot showing expression of mmp9:Citrine in myeloid cells (combined gates of Q1, Q2, Q3; Minimum = 12.4%, Maximum = 17.1%, Median = 15.7%; Box 25th−75th percentile). Violin plots indicating the distribution and expression levels of mmp9:Citrine among myeloid populations (mmp9:Citrine⁺ gate; n = 5 with pools of 15 triple transgenic larvae each). mfi = mean fluorescence intensity (d) mmp9:Citrine⁺ neutrophils (red histogram) have a higher side scatter (SSC) and therefore granularity compared to mmp9:Citrine^- neutrophils (blue histogram). Flow cytometric analysis of cells isolated from Tg(lysC:CFP-NTR)^vi002/ Tg(BACmmp9:Citrine-CAAX)^vi003 at 2 dpf. Cells were gated on the lysC:CFP⁺/ mmp9:Citrine⁺ or lysC:CFP⁺/ mmp9:Citrine^- populations and analyzed for SSC (n = 3). e Cells were isolated from kidneys, spleen or blood of adult Tg(lysC:dsRed)^nz50Tg/ Tg(BACmmp9:Citrine-CAAX)^vi003 and analyzed by flow cytometry for frequency of Mmp9⁺ cells. f Neutrophils were isolated from whole kidney marrow (WKM) of adult Tg(lysC:dsRed)^nz50Tg/ Tg(BACmmp9:Citrine-CAAX)^vi003 zebrafish and sorted into lysC:dsRed⁺/ mmp9:Citrine⁺or lysC:dsRed⁺/ mmp9:Citrine^- populations, cytospins were prepared and Pappenheim stained. Different neutrophil maturation stages were scored blinded, showing that the lysC:dsRed⁺/ mmp9:Citrine⁺ population consists of highly differentiated neutrophil stages. INT = intermediate; HI = high. Graph presents average percentages of n = 4 kidneys (721 Mmp9⁺ cells and 819 Mmp9^- cells were analyzed in total). g Representative ultrastructural images show an unsegmented nucleus and round and elongated granules in lysC:CFP⁺/ mmp9:Citrine^- cells (n = 5 from one TEM transmission electron microscopy experiment) contrasting with a segmented nucleus and predominantly elongated granules in lysC:CFP⁺/ mmp9:Citrine^HIcells (n = 2); *, round granules; #, elongated granules.