Detecting apoptosis of leukocytes in mouse lymph nodes

Gómez-Cabañas, Laura; Delgado-Martín, Cristina; López-Cotarelo, Pilar; Escribano-Díaz, Cristina; Alonso-C, Luis M; Riol-Blanco, Lorena; Rodrı́guez-Fernández, José Luis

doi:10.1038/nprot.2014.078

Cited by 9 publications

(9 citation statements)

References 46 publications

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“…After an additional 4.5 h, the animals were injected intravenously with SR-FLIVO, a poly-caspase binding inhibitor probe (VAD-FMK) conjugated to a fluorescent dye that binds irreversibly to apoptotic caspases and allows the detection of apoptotic cells in vivo (10,25,26,31). 1 h after the injection of SR-FLIVO, the mice were sacrificed, and the PLNs were obtained and analyzed by two-photon microscopy.…”

Section: Journal Of Biological Chemistrymentioning

confidence: 99%

“…Two-photon Microscopy and Analysis of Apoptotic CFSE-labeled Dendritic Cells in the Lymph Nodes-The method has been described in detail previously (10,25,26,31). Briefly, CFSE-labeled splenic mDCs (10 8 mDCs/ml) were dissolved in 20 l of RPMI and injected subcutaneously along with LPS (1 g/ml) into the hind footpad of recipient C57BL/6 mice (2 ϫ 10 6 mDCs per footpad).…”

Section: Purification Of Murine Dcs and Labeling Of The Cells With Flmentioning

confidence: 99%

“…40.5 h after initiation of the experiment, the mice were injected intravenously with SR-FLIVO (SR, abs 565 nm; em Ͼ600 nm), and after an additional 1 h, the mice were sacrificed, and the popliteal LNs were extracted from the mice and subsequently subjected to twophoton confocal analysis to visualize among the injected CFSEmDCs those that present SR-FLIVO staining (see "Results"). Two-photon microscopy and analysis of apoptotic CFSE-DCs in the LNs was performed as described previously (31).…”

Section: Purification Of Murine Dcs and Labeling Of The Cells With Flmentioning

confidence: 99%

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A Novel MEK-ERK-AMPK Signaling Axis Controls Chemokine Receptor CCR7-dependent Survival in Human Mature Dendritic Cells

López-Cotarelo

Escribano-Díaz

González-Bethencourt

et al. 2015

Journal of Biological Chemistry

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Background: Chemokine receptor CCR7 promotes survival in mature dendritic cells (mDCs). Results: Activated AMP-dependent kinase (AMPK) induces apoptosis in mDCs; CCR7 uses the kinases MEK1/2-ERK1/2 to regulate phosphorylation of AMPK on Ser-485 and consequently its inhibition. Conclusion: CCR7 uses a novel MEK1/2-ERK1/2-AMPK signaling axis to induce survival in mDCs. Significance: AMPK is a potential target to regulate mDC-mediated immune responses.

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Section: Journal Of Biological Chemistrymentioning

confidence: 99%

Section: Purification Of Murine Dcs and Labeling Of The Cells With Flmentioning

confidence: 99%

Section: Purification Of Murine Dcs and Labeling Of The Cells With Flmentioning

confidence: 99%

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A Novel MEK-ERK-AMPK Signaling Axis Controls Chemokine Receptor CCR7-dependent Survival in Human Mature Dendritic Cells

López-Cotarelo

Escribano-Díaz

González-Bethencourt

et al. 2015

Journal of Biological Chemistry

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“…In the experiments of localization of the mitochondria, the DCs were labeled for 30 min at 37˚C with MitoTracker Red (200 nM) in RPMI 1640 medium. IS formation was induced as indicated before (8,23). Briefly, first the DCs were pulsed in RPMI 1640 medium for 30 min with the specific Ag OVA 3232339 peptide or, in the case of the controls, with either of two nonspecific Ags, namely, human citrullinated vimentin peptide or human citrullinated fibrinogen.…”

Section: Purification Of Dcs and Cd4 T Cells Labeling Of The Cells Wmentioning

confidence: 99%

Immunological Synapse Formation Induces Mitochondrial Clustering and Mitophagy in Dendritic Cells

Gómez-Cabañas

López-Cotarelo

Criado‐García

et al. 2019

The Journal of Immunology

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The immunological synapse (IS) is a superstructure formed during T cell activation at the zone of contact between T cells and dendritic cells (DCs). The IS includes specific molecular components in the T cell and DCs sides that may result in different functionality. Most of the studies on the IS have focused on the T cell side of this structure and, in contrast, the information available on the IS of DCs is sparse. Autophagy is a cellular process involved in the clearance of damaged proteins and organelles via lysosomal degradation. Mitophagy is the selective autophagy of damaged mitochondria. In this study, it is shown that IS formation induces clustering of mitochondria in the IS of DCs and partial depolarization of these organelles. At the IS of the DCs also accumulate autophagy and mitophagy markers, even when the kinase complex mTORC1, an inhibitor of the autophagy, is active. Together the results presented indicate that IS formation induces local clustering of mitochondria and mitophagy, which could be a homeostatic mechanism to control the quality of mitochondria in this region. The data underline the complexity of the regulatory mechanisms operating in the IS of DCs.

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“…The cells were then fixed with cold methanol, and apoptotic nuclear morphology was assessed using Hoechst33342 staining as indicated previously (3,5,(26)(27)(28).…”

Section: Assays Of Apoptotic Damagementioning

confidence: 99%

The Mammalian Sterile 20–like 1 Kinase Controls Selective CCR7-Dependent Functions in Human Dendritic Cells

Torres‐Bacete

Delgado-Martín

Gómez-Moreira

et al. 2015

The Journal of Immunology

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The chemokine receptor CCR7 directs mature dendritic cells (mDCs) to the lymph nodes where these cells control the initiation of the immune response. CCR7 regulates chemotaxis, endocytosis, survival, migratory speed, and cytoarchitecture in mDCs. The molecular mechanisms used by CCR7 to regulate these functions in mDCs are not completely understood. The mammalian sterile 20–like 1 kinase (Mst1) plays a proapoptotic role under stress conditions; however, recently, it has been shown that Mst1 can also control homeostatic cell functions under normal conditions. In this study, we show that stimulation of CCR7 in mDCs induces Gαi-dependent activation of Mst1, suggesting the involvement of this kinase in the control of CCR7-dependent functions. Analysis of the mDCs in which Mst1 expression levels were reduced with small interfering RNA shows that this kinase mediates CCR7-dependent effects on cytoarchitecture, endocytosis and migratory speed but not on chemotaxis or survival. In line with these results, biochemical analysis indicates that Mst1 does not control key signaling regulators of CCR7-dependent chemotaxis or survival. In contrast, Mst1 regulates downstream of CCR7 and, of note, independently of Gα13, the RhoA pathway. Reduction of Mst1 inhibits CCR7-dependent phosphorylation of downstream targets of RhoA, including cofilin, myosin L chain, and myosin L chain phosphatase. Consistent with the role of the latter molecules as modulators of the actin cytoskeleton, mDCs with reduced Mst1 also displayed a dramatic reduction in actin barbed-end formation that could not be recovered by stimulating CCR7. The results indicate that the kinase Mst1 controls selective CCR7-dependent functions in human mDCs.

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Detecting apoptosis of leukocytes in mouse lymph nodes

Cited by 9 publications

References 46 publications

A Novel MEK-ERK-AMPK Signaling Axis Controls Chemokine Receptor CCR7-dependent Survival in Human Mature Dendritic Cells

A Novel MEK-ERK-AMPK Signaling Axis Controls Chemokine Receptor CCR7-dependent Survival in Human Mature Dendritic Cells

Immunological Synapse Formation Induces Mitochondrial Clustering and Mitophagy in Dendritic Cells

The Mammalian Sterile 20–like 1 Kinase Controls Selective CCR7-Dependent Functions in Human Dendritic Cells

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