( B) Graph represents the kinetic of the frequency of mpeg1 + tnfa + macrophages in macrophage population ( mpeg1 +) in three independent experiments following stimulation: amputation and E. Red, green, and yellow gates represent mCherry +, eGFP +, and mCherry +eGFP + populations, respectively. coli at 3 dpf, and cells were collected at 6 hr post-treatment. Tg(mpeg1:mCherryF/tnfa:eGFP-F) larvae were either kept intact (control), or amputated, or injected with PBS or with E. ( A) Graphed data of representative fluorescence-activated flow cytometry analysis of tnfa + and tnfa − macrophages upon inflammatory stimulations. Macrophages (red) barely establish cell–cell contact. ( J) Representative time-lapse maximum projections show the behaviour of tnfa − mpeg1 + macrophages, starting 25h12 pA during 52 min. ![]() These macrophages (eGFP in grey) remain attached up to 40 min. Two macrophages (green + red) interact by cell–cell contact. ( I) Representative time-lapse maximum projections show the behaviour of tnfa + mpeg1 + macrophages, starting 19h20 pA during 42 min. Measurements were extracted from three independent videos of amputated Tg(mpeg1:mCherryF/tnfa:eGFP-F), for contact frequency, N = 15 and for duration of the interaction, N = 11 macrophages. ( G) Frequency of macrophage–macrophage contacts and ( H) time length of the contacts of tnfa − mpeg1 + and tnfa + mpeg1 + cells. ( F) Velocity of tnfa + mpeg1 + and tnfa − mpeg1 + macrophages (N = 18). tnfa + mpeg1 + macrophages exhibit a round and protrusive morphology, while tnfa − mpeg1 + macrophages exhibit a dendritic morphology. ( D, E) Maximum projections of confocal analysis of eGFP-F (green) and mCherryF (red) expressions in recruited macrophages at ( D) 18 hpA and ( E) 24 hpA in Tg(mpeg1:mCherryF/tnfa:eGFP-F). The transcriptional activation of tnfa (green) in recruited macrophage (red, arrowhead) was first observed from 3 hpA. The time pA is shown on top right corner and indicated in hours and minutes, white lines outline the caudal fin. ( C) Representative time-lapse maximum projections show the activation of macrophages arriving at the wound in 3 dpf amputated Tg(mpeg1:mCherryF/tnfa:eGFP-F). Dotted red box shows the region imaged in C. ( B) Bright-field image of the wounded fin of a 3 dpf Tg(mpeg1:mCherryF/tnfa:eGFP-F) larva. Dotted lines outline the caudal fin, scale bar = 100 μm. Arrowheads show recruited macrophages that express tnfa, arrows show tnfa + cells that are not macrophages, and asterisks show auto-fluorescent pigments. ( A) eGFP-F (green) and mCherryF (red) fluorescence was analyzed by fluorescent microscopy in intact (control) and amputated Tg(mpeg1:mCherryF/tnfa:eGFP-F) fins at 6 hpA of 3 dpf larvae. Graph represents the mean value of three independent experiments ±s.e.m. Real-time RT-PCR on separated cells using EF1a as a reference gene. ( M) Relative expression of tnfa in eGFP- and GFP + cells in amputated larvae. Green gates represent eGFP + population and mean percentage of eGFP + population ±s.e.m is indicated. Tg(tnfa:eGFP-F) larvae were either kept intact (control) or amputated at 3 dpf, and cells were collected at 6 hr post-treatment. ( L) Graphed data of representative fluorescence-activated flow cytometry analysis of eGFP + cells in upon amputation. Dotted lines delimit the caudal fin, arrowheads show overlapping signals, and arrows show the pigments. ( K) tnfa mRNA and eGFP-F expressions were analyzed using microscopy at 6 hpA in amputated fins from 3 dpf Tg(tnfa:eGFP-F) larvae. coli-infected Tg(tnfa:eGFP-F) larvae, scale bar = 20 μm. ( J) Multi-scan confocal analysis of GFP expression in E. ![]() Arrows show auto-fluorescent xanthophores. ![]() ![]() ( F, G) eGFP fluorescence (green) was analyzed by fluorescent microscopy in ( F) intact (control) and ( G) amputated Tg(tnfa:eGFP-F) fins at 6 hpA, dotted lines outline the caudal fin, scale bar = 100 μm and at 16 hpi in Tg(tnfa:eGFP-F) larvae injected with ( H) PBS or ( I, J) E. ( E) Imaging of tnfa mRNA expression in the muscle at higher magnification, asterisks show muscle fibres, scale bar in ( B) = 100 μm and in ( E) = 50 μm. ( A– E) Tumour necrosis factor alpha ( tnfa) mRNA expression (blue, arrowhead) was detected by in situ hybridization using tnfa anti-sense probe: at 6 hpA in ( A) intact (control) and ( B) amputated fins from 3 dpf WT larvae, ( C) in uninfected larvae (54 hpf, hours post-fertilization) and ( D, E) E.
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