Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo methods
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Innate immune cells are recruited to the vessel wall early during DVT formation
reagent used in the protocol.
- Use
- Neutrophils and monocytes are the main leukocyte subsets accumulating during the initiation of DVT. (A) Neutrophils (green) crawling on the vessel wall (red) of the IVC 2 h after DVT induction visualized by two-photon microscopy. Tracks of individual neutrophils are shown in white (also see Video 3 ). Bar, 50 µ...
Innate immune cells are recruited to the vessel wall early during DVT formation
reagent used in the protocol.
- Use
- To dissect the leukocyte subsets recruited in response to depressed blood flow, we next induced flow restriction in LysM-eGFP reporter mice. We observed that >80% of all cells recruited during the first 6 h were eGFP hi ( ). In addition to neutrophils, monocytes also express eGFP in LysM-eGFP mutants, albeit at lowe...
Blood cell-derived TF is a central initiator of DVT
reagent used in the protocol.
- Use
- Blood cell TF is indispensable for venous thrombosis. (A) Fibrin formation during DVT development was measured in vivo by intravital microscopy in control HCV and low-hTF mice using an Alexa Fluor 488-labeled specific fibrin antibody. Measurements were performed after 1-6 h of flow restriction. Represent...
Neutrophils promote propagation of DVT via NET formation
reagent used in the protocol.
- Use
- Given their frequency among leukocytes recruited to the IVC, we next examined the contribution of neutrophils to DVT development in more detail. To address this, we depleted neutrophils by injection of an anti-Ly6G antibody and induced flow restriction in the IVC ( and not depicted). To our surprise, neutropenic mic...
Neutrophils promote propagation of DVT via NET formation
reagent used in the protocol.
- Use
- NETs propagate DVT in vivo. (A) Leukocyte accumulation in vivo at 6 h of flow restriction in the IVC of LysM-eGFP mice treated with control antibody or the anti-Ly6G mAb to deplete neutrophils. Arrowhead: aggregated neutrophils; arrows: single, adherent cells. Bars, 100 µm. (B) Thrombus weight 48 h after DVT i...
Neutrophils promote propagation of DVT via NET formation
reagent used in the protocol.
- Use
- Because platelets and a dense network of fibrin/fibrinogen surrounded the NETs, we analyzed in more detail whether NETs concentrate prothrombotic factors on their surfaces. In fact, the NETs were decorated with TF and protein disulfide isomerase, an enzyme implicated in the activation of blood cell-derived TF,...
Platelets support DVT formation
reagent used in the protocol.
- Use
- Platelet recruitment supports DVT formation in vivo. (A) Immunohistological cross sections of the IVC 48 h after DVT induction display platelet accumulation (CD41 + ) within the thrombus. Nuclei are counterstained with DAPI. Bars, 200 µm. Representative of n = 3 experiments. (B) Representative images of intravi...
Platelets support DVT formation
reagent used in the protocol.
- Use
- To evaluate the quantitative contribution of platelets to the cellular content of the thrombus, we stained 100% of platelets by injecting a fluorescently labeled anti-GPIbβ antibody, which does not interfere with platelet adhesion and aggregation. After 6 h of flow reduction, platelets were diffusely distribute...
Innate immune cells are recruited to the vessel wall early during DVT formation
Although recent studies have suggested a link between inflammation and DVT, the precise role of immune cells in DVT development remains unclear (; ). When we examined venous thrombi that developed in response to 48 h of flow restriction, inflammatory leukocytes were in fact a major cellular constituent. Leukocytes...
- Use
- Although recent studies have suggested a link between inflammation and DVT, the precise role of immune cells in DVT development remains unclear (; ). When we examined venous thrombi that developed in response to 48 h of flow restriction, inflammatory leukocytes were in fact a major cellular constituent. Leukocytes...
Innate immune cells are recruited to the vessel wall early during DVT formation
We then determined whether accumulation of innate immune cells is a consequence or rather a cause of DVT formation. Scanning and transmission electron microscopy, as well as standard histology, revealed that leukocytes were recruited to the IVC in large quantities already after 6 h of flow restriction ( ). Leukocyte...
- Use
- We then determined whether accumulation of innate immune cells is a consequence or rather a cause of DVT formation. Scanning and transmission electron microscopy, as well as standard histology, revealed that leukocytes were recruited to the IVC in large quantities already after 6 h of flow restriction ( ). Leukocyte...
Innate immune cells are recruited to the vessel wall early during DVT formation
Neutrophils and monocytes are the main leukocyte subsets accumulating during the initiation of DVT. (A) Neutrophils (green) crawling on the vessel wall (red) of the IVC 2 h after DVT induction visualized by two-photon microscopy. Tracks of individual neutrophils are shown in white (also see Video 3 ). Bar, 50 µ...
- Use
- Neutrophils and monocytes are the main leukocyte subsets accumulating during the initiation of DVT. (A) Neutrophils (green) crawling on the vessel wall (red) of the IVC 2 h after DVT induction visualized by two-photon microscopy. Tracks of individual neutrophils are shown in white (also see Video 3 ). Bar, 50 µ...
Innate immune cells are recruited to the vessel wall early during DVT formation
To dissect the leukocyte subsets recruited in response to depressed blood flow, we next induced flow restriction in LysM-eGFP reporter mice. We observed that >80% of all cells recruited during the first 6 h were eGFP hi ( ). In addition to neutrophils, monocytes also express eGFP in LysM-eGFP mutants, albeit at lowe...
- Use
- To dissect the leukocyte subsets recruited in response to depressed blood flow, we next induced flow restriction in LysM-eGFP reporter mice. We observed that >80% of all cells recruited during the first 6 h were eGFP hi ( ). In addition to neutrophils, monocytes also express eGFP in LysM-eGFP mutants, albeit at lowe...
Leukocyte accumulation depends on endothelial P-selectin
Crucial role of P-selectin for leukocyte accumulation in DVT. (A) RT-PCR of trafficking molecules in the IVC in response to flow restriction at baseline or 6 and 48 h after DVT induction ( n = 5 per group). Results are shown as mean ± standard deviation. (B) RT-PCR of P-selectin in the IVC at baseline or 6 and...
- Use
- Crucial role of P-selectin for leukocyte accumulation in DVT. (A) RT-PCR of trafficking molecules in the IVC in response to flow restriction at baseline or 6 and 48 h after DVT induction ( n = 5 per group). Results are shown as mean ± standard deviation. (B) RT-PCR of P-selectin in the IVC at baseline or 6 and...
Neutrophils promote propagation of DVT via NET formation
NETs propagate DVT in vivo. (A) Leukocyte accumulation in vivo at 6 h of flow restriction in the IVC of LysM-eGFP mice treated with control antibody or the anti-Ly6G mAb to deplete neutrophils. Arrowhead: aggregated neutrophils; arrows: single, adherent cells. Bars, 100 µm. (B) Thrombus weight 48 h after DVT i...
- Use
- NETs propagate DVT in vivo. (A) Leukocyte accumulation in vivo at 6 h of flow restriction in the IVC of LysM-eGFP mice treated with control antibody or the anti-Ly6G mAb to deplete neutrophils. Arrowhead: aggregated neutrophils; arrows: single, adherent cells. Bars, 100 µm. (B) Thrombus weight 48 h after DVT i...
Neutrophils promote propagation of DVT via NET formation
Because TF expression by Ly6G + neutrophils was weak compared with monocytes ( ), it seemed likely that, apart from acting as a potential source of myeloid TF, neutrophils deliver additional signals supporting venous thrombogenesis. Recently, activated neutrophils have been shown to release NETs, which consist of ex...
- Use
- Because TF expression by Ly6G + neutrophils was weak compared with monocytes ( ), it seemed likely that, apart from acting as a potential source of myeloid TF, neutrophils deliver additional signals supporting venous thrombogenesis. Recently, activated neutrophils have been shown to release NETs, which consist of ex...
Platelets support DVT formation
Platelet recruitment supports DVT formation in vivo. (A) Immunohistological cross sections of the IVC 48 h after DVT induction display platelet accumulation (CD41 + ) within the thrombus. Nuclei are counterstained with DAPI. Bars, 200 µm. Representative of n = 3 experiments. (B) Representative images of intravi...
- Use
- Platelet recruitment supports DVT formation in vivo. (A) Immunohistological cross sections of the IVC 48 h after DVT induction display platelet accumulation (CD41 + ) within the thrombus. Nuclei are counterstained with DAPI. Bars, 200 µm. Representative of n = 3 experiments. (B) Representative images of intravi...
Statistical analysis.
Software used for acquisition, scoring, statistics, or reporting.
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- All data are shown as mean ± SEM, unless indicated otherwise. Thrombus weight was tested for normal distribution using the Kolmogorov-Smirnov test and the independent samples Student's t test was performed to compare groups (SPSS). More than two groups were compared using the ANOVA-LSD post hoc test. Para...
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Innate immune cells are recruited to the vessel wall early during DVT formation
To dissect the leukocyte subsets recruited in response to depressed blood flow, we next induced flow restriction in LysM-eGFP reporter mice. We observed that >80% of all cells recruited during the first 6 h were eGFP hi ( ). In addition to neutrophils, monocytes also express eGFP in LysM-eGFP mutants, albeit at lower levels that are beyond the detection limit of 2P-IVM ( ). Correspondingly, 2P-IVM revealed that ∼90% of all eGFP hi cells recruited to venous thrombi also stain positive for Ly6G, indicating that eGFP hi cells indeed mainly represent neutrophils ( and Video 4 ). We then examined heterozygous CX 3 CR1-eGFP mice to specifically define monocyte recruitment. This revealed that CX 3 CR1 + monocytes constitute ∼15% of the recruited leukocytes (; and Video 5 ). Together, these findings indicate that neutrophils and monocytes are the predominant leukocyte subsets tha...
Leukocyte accumulation depends on endothelial P-selectin
Crucial role of P-selectin for leukocyte accumulation in DVT. (A) RT-PCR of trafficking molecules in the IVC in response to flow restriction at baseline or 6 and 48 h after DVT induction ( n = 5 per group). Results are shown as mean ± standard deviation. (B) RT-PCR of P-selectin in the IVC at baseline or 6 and 48 h after DVT induction ( n = 5 per group). Results are shown as mean ± standard deviation. (C) Representative immunohistochemical stainings of the IVC endothelium 48 h after DVT induction showing P-selectin and vWF on the endothelial surface. Nuclei are counterstained with DAPI. Bars, 50 µm. (D, Left) Representative in vivo images of adherent leukocytes in C57BL/6 and SELP -/- mice 6 h after induction of DVT. Leukocytes were stained with Acridine orange and visualized by intravital video microscopy (arrowhead indicates aggregates; arrows indicate sin...
Platelets support DVT formation
Platelet recruitment supports DVT formation in vivo. (A) Immunohistological cross sections of the IVC 48 h after DVT induction display platelet accumulation (CD41 + ) within the thrombus. Nuclei are counterstained with DAPI. Bars, 200 µm. Representative of n = 3 experiments. (B) Representative images of intravital video microscopy of blood cell recruitment taken at 6 h after DVT induction. Arrowheads: thrombi; arrows: single, adherent cells. Platelets, red (rhodamine B); leukocytes, green (Acridine orange). Bars, 100 µm. (C) Time-lapse images of the developing thrombus (arrowheads) visualized by two-photon microscopy 6 h after DVT induction. Platelets (yellow) and neutrophils (green) are recruited from the bloodstream (blue) to the vessel wall (red; see also Video 8 ). (D) Platelet-leukocyte interaction was determined by intravital microscopy in C57BL/6 and IL4-R/Iba m...
Mouse model of flow restriction in the IVC.
Mice were anesthetized by intraperitoneal injection as described previously ( ). A median laparotomy was performed and the IVC was exposed by atraumatic surgery. We positioned a space holder (FloppyR II Guide Wire 0.014 in [0.36 mm]; Guidant Corporation) on the outside of the vessel and we placed a permanent narrowing ligature (8.0 monofil polypropylene filament, Premilene; Braun) exactly below the left renal vein. Subsequently, the wire was removed to avoid complete vessel occlusion. Side branches were not ligated or manipulated. Flow velocity was determined immediately after the flow restriction (Cap-Image 7.1). Because we wanted to rule out endothelial injury as a trigger for venous thrombosis, all mice with bleedings or any injury of the IVC during surgery were excluded from further analysis. There was no difference in the exclusion rate across the different experimental groups. A...
Preparation of platelets and leukocytes for intravital microscopy.
Murine platelets were isolated from whole blood and labeled with 5-carboxyflourescein diacetate succinimidyl ester (DCF) or cell tracker violet (Invitrogen) as reported earlier ( ). The DCF-labeled platelet suspension was adjusted to a final concentration of 150 × 10 6 platelets/250 µl and injected i.v. via a jugular vein catheter. For in vivo staining, a nonblocking GPIbβ-binding fluorescent-labeled antibody (3 µg/animal; rat anti-mouse DyLight488-labeled GPIbβ antibody; Emfret Analytics) was infused i.v. Adhesion and aggregation of murine platelets were assessed by in vivo video microscopy. For the qualification and quantification of leukocyte adhesion, rhodamine 6G (Invitrogen) or Acridine orange (Sigma-Aldrich) was injected i.v. to stain circulating leukocytes in vivo. To characterize platelet-leukocyte interactions in vivo, Acridine orange-sta...
Assessment of fibrin formation in vivo.
Fibrin formation in vivo was measured in HCV and Low-hTF using an Alexa Flour 488 (Invitrogen) ex vivo labeled anti-fibrin antibody (2 mg/kg body weight, mouse anti-fibrin IIβ chain Bβ 15-42, clone T2G1; Accurate Chemical) which was injected i.v. This antibody specifically binds to fibrin formed in the vascular system. Fluorescence intensity was quantified by intravital video microscopy (BX51WI; Olympus). Fibrin formation is presented as mean fluorescence intensity (arbitrary units).
Intravital epifluorescence microscopy.
The mice were anesthetized as described, the flow restricting procedure was performed, and the animals were fixed on a custom built-stage to maintain a physiological temperature. Measurements were performed with a high-speed wide-field Olympus BX51WI fluorescence microscope using a long-distance condenser and a 20 × (NA 0.95) water immersion objective with a monochromator (MT 20; Olympus) and a charge-coupled device camera (ORCA-ER; Hamamatsu Photonics). For image acquisition and analysis a computer (Dell) with Cell^R (Olympus) software was used. Cell recruitment was quantified in four fields of view (50 × 100 µm) per animal; immotile cells were counted as adherent and moving cells were counted as rolling within 30 s as previously described ( ). The cell covered area (in square millimeters) and colocalization area was determined by planimetric measurement (Cap-Image 7.1).
Two-photon in vivo microscopy.
For intravital imaging of the IVC in vivo, the TrimScope (LaVision Biotech) connected to an upright microscope (Olympus) was used, equipped with a MaiTai laser (Spectra-Physics) and a 20 × 0.95 NA water immersion objective (Olympus). Pictures were acquired at 800 nm excitation wavelength in a 500 × 500-µm frame with 512 × 512 pixels and a z-step of 3 µm and detected by PMTs (G6780-20, Hamamatsu). ImSpector (LaVision Biotech) was used as acquisition software. The mice were anesthetized as described, the flow restricting procedure was performed, and the animals were fixed on a custom-built stage to maintain a physiological temperature. LysM-eGFP mice, CX 3 CR1-eGFP mice, and an FITC- or PE-labeled anti-Ly6G antibody (10 µg/animal; Clone 1A8; eBioscience) were used to visualize neutrophils and monocytes. In addition, cell tracker violet (Invitrogen)-lab...
Measurement outputs
What raw and processed outputs should exist?
Leukocytes are recruited during the early phase of venous thrombosis to the intact endothelial surface. (A) Leukocyte accumulation in DVT induced by 48 h of flow restriction. vG...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Neutrophils and monocytes are the main leukocyte subsets accumulating during the initiation of DVT. (A) Neutrophils (green) crawling on the vessel wall (red) of the IVC 2 h afte...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Crucial role of P-selectin for leukocyte accumulation in DVT. (A) RT-PCR of trafficking molecules in the IVC in response to flow restriction at baseline or 6 and 48 h after DVT...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Blood cell TF is indispensable for venous thrombosis. (A) Fibrin formation during DVT development was measured in vivo by intravital microscopy in control HCV and low-hTF mice u...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
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inferred from protocolPreprocessing / cleaning
Leukocytes are recruited during the early phase of venous thrombosis to the intact endothelial surface.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Leukocytes are recruited during the early phase of venous thrombosis to the intact endothelial surface. (A) Leukocyte accumulation in DVT induced by 48 h of flow restriction. vG...; Neutrophils and monocytes are the main leukocyte subsets accumulating during the initiation of DVT. (A) Neutrophils (green) crawling on the vessel wall (red) of the IVC 2 h afte...; Crucial role of P-selectin for leukocyte accumulation in DVT. (A) RT-PCR of trafficking molecules in the IVC in response to flow restriction at baseline or 6 and 48 h after DVT...; Blood cell TF is indispensable for venous thrombosis. (A) Fibrin formation during DVT development was measured in vivo by intravital microscopy in control HCV and low-hTF mice u....
from paperStatistical comparison
Leukocytes are recruited during the early phase of venous thrombosis to the intact endothelial surface. (A) Leukocyte accumulation in DVT induced by 48 h of flow restriction. vG...; Neutrophils and monocytes are the main leukocyte subsets accumulating during the initiation of DVT. (A) Neutrophils (green) crawling on the vessel wall (red) of the IVC 2 h afte...; Crucial role of P-selectin for leukocyte accumulation in DVT. (A) RT-PCR of trafficking molecules in the IVC in response to flow restriction at baseline or 6 and 48 h after DVT...; Blood cell TF is indispensable for venous thrombosis. (A) Fibrin formation during DVT development was measured in vivo by intravital microscopy in control HCV and low-hTF mice u...
from paperReporting output
Report representative outputs alongside summary comparisons for Leukocytes are recruited during the early phase of venous thrombosis to the intact endothelial surface. (A) Leukocyte accumulation in DVT induced by 48 h of flow restriction. vG..., Neutrophils and monocytes are the main leukocyte subsets accumulating during the initiation of DVT. (A) Neutrophils (green) crawling on the vessel wall (red) of the IVC 2 h afte..., Crucial role of P-selectin for leukocyte accumulation in DVT. (A) RT-PCR of trafficking molecules in the IVC in response to flow restriction at baseline or 6 and 48 h after DVT..., Blood cell TF is indispensable for venous thrombosis. (A) Fibrin formation during DVT development was measured in vivo by intravital microscopy in control HCV and low-hTF mice u....
inferred from protocolStructured statistical methods
Leukocytes are recruited during the early phase of venous thrombosis to the intact endothelial surface. (A) Leukocyte accumulation in DVT induced by 48 h of flow restriction. vG...; Neutrophils and monocytes are the main leukocyte subsets accumulating during the initiation of DVT. (A) Neutrophils (green) crawling on the vessel wall (red) of the IVC 2 h afte...; Crucial role of P-selectin for leukocyte accumulation in DVT. (A) RT-PCR of trafficking molecules in the IVC in response to flow restriction at baseline or 6 and 48 h after DVT...; Blood cell TF is indispensable for venous thrombosis. (A) Fibrin formation during DVT development was measured in vivo by intravital microscopy in control HCV and low-hTF mice u...
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Evidence quotes (8)
To dissect the leukocyte subsets recruited in response to depressed blood flow, we next induced flow restriction in LysM-eGFP reporter mice. We observed that >80% of all cells recruited during the first 6 h were eGFP hi ( ). In addition to neutrophils, monocytes also express eGFP in LysM-eGFP mutants, albeit at lower levels that are beyond the detection limit of 2P-IVM ( ). Correspondingly, 2P-IVM revealed that ∼90% of all eGFP hi cells recruited to venous thrombi also stain positive for Ly6G, indicating that eGFP hi cells indeed mainly represent neutrophils ( and Video 4 ). We then examined heterozygous CX 3 CR1-eGFP mice to specifically define monocyte recruitment. This revealed that CX 3 CR1 + monocytes constitute ∼15% of the recruited leukocytes (; and Video 5 ). Together, these findings indicate that neutrophils and monocytes are the predominant leukocyte subsets that actively accumulate at the vascular surface during DVT development.
Crucial role of P-selectin for leukocyte accumulation in DVT. (A) RT-PCR of trafficking molecules in the IVC in response to flow restriction at baseline or 6 and 48 h after DVT induction ( n = 5 per group). Results are shown as mean ± standard deviation. (B) RT-PCR of P-selectin in the IVC at baseline or 6 and 48 h after DVT induction ( n = 5 per group). Results are shown as mean ± standard deviation. (C) Representative immunohistochemical stainings of the IVC endothelium 48 h after DVT induction showing P-selectin and vWF on the endothelial surface. Nuclei are counterstained with DAPI. Bars, 50 µm. (D, Left) Representative in vivo images of adherent leukocytes in C57BL/6 and SELP -/- mice 6 h after induction of DVT. Leukocytes were stained with Acridine orange and visualized by intravital video microscopy (arrowhead indicates aggregates; arrows indicate single adherent cells). Bars, 100 µm. (D, Right) Quantitative analysis of firm leukocyte adhesion, 6 h after flow restriction. Firm cell adhesion is given in number per square millimeters of C57BL/6 ( n = 5) and SELP -/- ( n = 7). Data are shown as mean ± SEM. (E, Left) Representa...
Platelet recruitment supports DVT formation in vivo. (A) Immunohistological cross sections of the IVC 48 h after DVT induction display platelet accumulation (CD41 + ) within the thrombus. Nuclei are counterstained with DAPI. Bars, 200 µm. Representative of n = 3 experiments. (B) Representative images of intravital video microscopy of blood cell recruitment taken at 6 h after DVT induction. Arrowheads: thrombi; arrows: single, adherent cells. Platelets, red (rhodamine B); leukocytes, green (Acridine orange). Bars, 100 µm. (C) Time-lapse images of the developing thrombus (arrowheads) visualized by two-photon microscopy 6 h after DVT induction. Platelets (yellow) and neutrophils (green) are recruited from the bloodstream (blue) to the vessel wall (red; see also Video 8 ). (D) Platelet-leukocyte interaction was determined by intravital microscopy in C57BL/6 and IL4-R/Iba mice after 6 h of flow restriction. Bars: (left) 50 µm; (right) 100 µm. The right panel shows quantification of colocalization of leukocytes and platelets in WT ( n = 5) and IL4-R/Iba mice ( n = 4). Data are shown as mean ± SEM. (E) Representative images obtained by video microscopy 6...
Mice were anesthetized by intraperitoneal injection as described previously ( ). A median laparotomy was performed and the IVC was exposed by atraumatic surgery. We positioned a space holder (FloppyR II Guide Wire 0.014 in [0.36 mm]; Guidant Corporation) on the outside of the vessel and we placed a permanent narrowing ligature (8.0 monofil polypropylene filament, Premilene; Braun) exactly below the left renal vein. Subsequently, the wire was removed to avoid complete vessel occlusion. Side branches were not ligated or manipulated. Flow velocity was determined immediately after the flow restriction (Cap-Image 7.1). Because we wanted to rule out endothelial injury as a trigger for venous thrombosis, all mice with bleedings or any injury of the IVC during surgery were excluded from further analysis. There was no difference in the exclusion rate across the different experimental groups. After the procedure, a subset of animals was investigated by intravital microscopy. In the remainder, the median laparotomy was immediately sutured by a 7.0 polypropylene suture (Ethicon). For weight measurement, the vessel was excised just below the renal veins and proximal to the confluence of the...
Murine platelets were isolated from whole blood and labeled with 5-carboxyflourescein diacetate succinimidyl ester (DCF) or cell tracker violet (Invitrogen) as reported earlier ( ). The DCF-labeled platelet suspension was adjusted to a final concentration of 150 × 10 6 platelets/250 µl and injected i.v. via a jugular vein catheter. For in vivo staining, a nonblocking GPIbβ-binding fluorescent-labeled antibody (3 µg/animal; rat anti-mouse DyLight488-labeled GPIbβ antibody; Emfret Analytics) was infused i.v. Adhesion and aggregation of murine platelets were assessed by in vivo video microscopy. For the qualification and quantification of leukocyte adhesion, rhodamine 6G (Invitrogen) or Acridine orange (Sigma-Aldrich) was injected i.v. to stain circulating leukocytes in vivo. To characterize platelet-leukocyte interactions in vivo, Acridine orange-stained leukocytes and ex vivo labeled platelets (20 µg/ml rhodamine B; Sigma-Aldrich) were imaged simultaneously. To differentiate neutrophils and monocytes, LysM-eGFP and CX3CR1-eGFP mice were used. The ratio of these cell types in relation to the total number of recruited leukocytes was analyze...
Fibrin formation in vivo was measured in HCV and Low-hTF using an Alexa Flour 488 (Invitrogen) ex vivo labeled anti-fibrin antibody (2 mg/kg body weight, mouse anti-fibrin IIβ chain Bβ 15-42, clone T2G1; Accurate Chemical) which was injected i.v. This antibody specifically binds to fibrin formed in the vascular system. Fluorescence intensity was quantified by intravital video microscopy (BX51WI; Olympus). Fibrin formation is presented as mean fluorescence intensity (arbitrary units).
The mice were anesthetized as described, the flow restricting procedure was performed, and the animals were fixed on a custom built-stage to maintain a physiological temperature. Measurements were performed with a high-speed wide-field Olympus BX51WI fluorescence microscope using a long-distance condenser and a 20 × (NA 0.95) water immersion objective with a monochromator (MT 20; Olympus) and a charge-coupled device camera (ORCA-ER; Hamamatsu Photonics). For image acquisition and analysis a computer (Dell) with Cell^R (Olympus) software was used. Cell recruitment was quantified in four fields of view (50 × 100 µm) per animal; immotile cells were counted as adherent and moving cells were counted as rolling within 30 s as previously described ( ). The cell covered area (in square millimeters) and colocalization area was determined by planimetric measurement (Cap-Image 7.1).
For intravital imaging of the IVC in vivo, the TrimScope (LaVision Biotech) connected to an upright microscope (Olympus) was used, equipped with a MaiTai laser (Spectra-Physics) and a 20 × 0.95 NA water immersion objective (Olympus). Pictures were acquired at 800 nm excitation wavelength in a 500 × 500-µm frame with 512 × 512 pixels and a z-step of 3 µm and detected by PMTs (G6780-20, Hamamatsu). ImSpector (LaVision Biotech) was used as acquisition software. The mice were anesthetized as described, the flow restricting procedure was performed, and the animals were fixed on a custom-built stage to maintain a physiological temperature. LysM-eGFP mice, CX 3 CR1-eGFP mice, and an FITC- or PE-labeled anti-Ly6G antibody (10 µg/animal; Clone 1A8; eBioscience) were used to visualize neutrophils and monocytes. In addition, cell tracker violet (Invitrogen)-labeled platelets and 2 MD TRITC-Dextran (Invitrogen) for visualization of the blood flow were infused. 3D reconstruction and volume rendering was done with by Volocity (PerkinElmer) and cell tracking with ImageJ software (National Institutes of Health).
Machine-readable layer
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"name": "Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo methods",
"description": "Evidence-backed execution summary for Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo methods from Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.",
"totalTime": "PT5M",
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{
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"position": 1,
"name": "Innate immune cells are recruited to the vessel wall early during DVT formation",
"text": "To dissect the leukocyte subsets recruited in response to depressed blood flow, we next induced flow restriction in LysM-eGFP reporter mice. We observed that >80% of all cells recruited during the first 6 h were eGFP hi ( ). In addition to neutrophils, monocytes also express eGFP in LysM-eGFP mutants, albeit at lower levels that are beyond the detection limit of 2P-IVM ( ). Correspondingly, 2P-IVM revealed that ∼90% of all eGFP hi cells recruited to venous thrombi also stain positive for Ly6G, indicating that eGFP hi cells indeed mainly represent neutrophils ( and Video 4 ). We then examined heterozygous CX 3 CR1-eGFP mice to specifically define monocyte recruitment. This revealed that CX 3 CR1 + monocytes constitute ∼15% of the recruited leukocytes (; and Video 5 ). Together, these findings indicate that neutrophils and monocytes are the predominant leukocyte subsets tha..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Leukocyte accumulation depends on endothelial P-selectin",
"text": "Crucial role of P-selectin for leukocyte accumulation in DVT. (A) RT-PCR of trafficking molecules in the IVC in response to flow restriction at baseline or 6 and 48 h after DVT induction ( n = 5 per group). Results are shown as mean ± standard deviation. (B) RT-PCR of P-selectin in the IVC at baseline or 6 and 48 h after DVT induction ( n = 5 per group). Results are shown as mean ± standard deviation. (C) Representative immunohistochemical stainings of the IVC endothelium 48 h after DVT induction showing P-selectin and vWF on the endothelial surface. Nuclei are counterstained with DAPI. Bars, 50 µm. (D, Left) Representative in vivo images of adherent leukocytes in C57BL/6 and SELP -/- mice 6 h after induction of DVT. Leukocytes were stained with Acridine orange and visualized by intravital video microscopy (arrowhead indicates aggregates; arrows indicate sin..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Platelets support DVT formation",
"text": "Platelet recruitment supports DVT formation in vivo. (A) Immunohistological cross sections of the IVC 48 h after DVT induction display platelet accumulation (CD41 + ) within the thrombus. Nuclei are counterstained with DAPI. Bars, 200 µm. Representative of n = 3 experiments. (B) Representative images of intravital video microscopy of blood cell recruitment taken at 6 h after DVT induction. Arrowheads: thrombi; arrows: single, adherent cells. Platelets, red (rhodamine B); leukocytes, green (Acridine orange). Bars, 100 µm. (C) Time-lapse images of the developing thrombus (arrowheads) visualized by two-photon microscopy 6 h after DVT induction. Platelets (yellow) and neutrophils (green) are recruited from the bloodstream (blue) to the vessel wall (red; see also Video 8 ). (D) Platelet-leukocyte interaction was determined by intravital microscopy in C57BL/6 and IL4-R/Iba m..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Mouse model of flow restriction in the IVC.",
"text": "Mice were anesthetized by intraperitoneal injection as described previously ( ). A median laparotomy was performed and the IVC was exposed by atraumatic surgery. We positioned a space holder (FloppyR II Guide Wire 0.014 in [0.36 mm]; Guidant Corporation) on the outside of the vessel and we placed a permanent narrowing ligature (8.0 monofil polypropylene filament, Premilene; Braun) exactly below the left renal vein. Subsequently, the wire was removed to avoid complete vessel occlusion. Side branches were not ligated or manipulated. Flow velocity was determined immediately after the flow restriction (Cap-Image 7.1). Because we wanted to rule out endothelial injury as a trigger for venous thrombosis, all mice with bleedings or any injury of the IVC during surgery were excluded from further analysis. There was no difference in the exclusion rate across the different experimental groups. A..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Preparation of platelets and leukocytes for intravital microscopy.",
"text": "Murine platelets were isolated from whole blood and labeled with 5-carboxyflourescein diacetate succinimidyl ester (DCF) or cell tracker violet (Invitrogen) as reported earlier ( ). The DCF-labeled platelet suspension was adjusted to a final concentration of 150 × 10 6 platelets/250 µl and injected i.v. via a jugular vein catheter. For in vivo staining, a nonblocking GPIbβ-binding fluorescent-labeled antibody (3 µg/animal; rat anti-mouse DyLight488-labeled GPIbβ antibody; Emfret Analytics) was infused i.v. Adhesion and aggregation of murine platelets were assessed by in vivo video microscopy. For the qualification and quantification of leukocyte adhesion, rhodamine 6G (Invitrogen) or Acridine orange (Sigma-Aldrich) was injected i.v. to stain circulating leukocytes in vivo. To characterize platelet-leukocyte interactions in vivo, Acridine orange-sta..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Assessment of fibrin formation in vivo.",
"text": "Fibrin formation in vivo was measured in HCV and Low-hTF using an Alexa Flour 488 (Invitrogen) ex vivo labeled anti-fibrin antibody (2 mg/kg body weight, mouse anti-fibrin IIβ chain Bβ 15-42, clone T2G1; Accurate Chemical) which was injected i.v. This antibody specifically binds to fibrin formed in the vascular system. Fluorescence intensity was quantified by intravital video microscopy (BX51WI; Olympus). Fibrin formation is presented as mean fluorescence intensity (arbitrary units)."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Intravital epifluorescence microscopy.",
"text": "The mice were anesthetized as described, the flow restricting procedure was performed, and the animals were fixed on a custom built-stage to maintain a physiological temperature. Measurements were performed with a high-speed wide-field Olympus BX51WI fluorescence microscope using a long-distance condenser and a 20 × (NA 0.95) water immersion objective with a monochromator (MT 20; Olympus) and a charge-coupled device camera (ORCA-ER; Hamamatsu Photonics). For image acquisition and analysis a computer (Dell) with Cell^R (Olympus) software was used. Cell recruitment was quantified in four fields of view (50 × 100 µm) per animal; immotile cells were counted as adherent and moving cells were counted as rolling within 30 s as previously described ( ). The cell covered area (in square millimeters) and colocalization area was determined by planimetric measurement (Cap-Image 7.1)."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Two-photon in vivo microscopy.",
"text": "For intravital imaging of the IVC in vivo, the TrimScope (LaVision Biotech) connected to an upright microscope (Olympus) was used, equipped with a MaiTai laser (Spectra-Physics) and a 20 × 0.95 NA water immersion objective (Olympus). Pictures were acquired at 800 nm excitation wavelength in a 500 × 500-µm frame with 512 × 512 pixels and a z-step of 3 µm and detected by PMTs (G6780-20, Hamamatsu). ImSpector (LaVision Biotech) was used as acquisition software. The mice were anesthetized as described, the flow restricting procedure was performed, and the animals were fixed on a custom-built stage to maintain a physiological temperature. LysM-eGFP mice, CX 3 CR1-eGFP mice, and an FITC- or PE-labeled anti-Ly6G antibody (10 µg/animal; Clone 1A8; eBioscience) were used to visualize neutrophils and monocytes. In addition, cell tracker violet (Invitrogen)-lab..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Innate immune cells are recruited to the vessel wall early during DVT formation"
},
{
"@type": "HowToTool",
"name": "Innate immune cells are recruited to the vessel wall early during DVT formation"
},
{
"@type": "HowToTool",
"name": "Innate immune cells are recruited to the vessel wall early during DVT formation"
},
{
"@type": "HowToTool",
"name": "Innate immune cells are recruited to the vessel wall early during DVT formation"
},
{
"@type": "HowToTool",
"name": "Leukocyte accumulation depends on endothelial P-selectin"
},
{
"@type": "HowToTool",
"name": "Neutrophils promote propagation of DVT via NET formation"
},
{
"@type": "HowToTool",
"name": "Neutrophils promote propagation of DVT via NET formation"
},
{
"@type": "HowToTool",
"name": "Platelets support DVT formation"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Innate immune cells are recruited to the vessel wall early during DVT formation"
},
{
"@type": "HowToSupply",
"name": "Innate immune cells are recruited to the vessel wall early during DVT formation"
},
{
"@type": "HowToSupply",
"name": "Blood cell-derived TF is a central initiator of DVT"
},
{
"@type": "HowToSupply",
"name": "Neutrophils promote propagation of DVT via NET formation"
},
{
"@type": "HowToSupply",
"name": "Neutrophils promote propagation of DVT via NET formation"
},
{
"@type": "HowToSupply",
"name": "Neutrophils promote propagation of DVT via NET formation"
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"name": "Platelets support DVT formation"
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