FlowMAb APC anti-mouse Ly6G
Product Details
The 1A8 monoclonal antibody reacts with mouse Ly6G. Ly6G is a 21-25 kDa member of the Ly-6 superfamily of GPI-anchored cell surface proteins with roles in cell signaling and cell adhesion. Ly6G is expressed differentially during development by cells in the myeloid lineage including monocytes, macrophages, granulocytes, and neutrophils. Monocytes typically express Ly6G transiently during development while mature granulocytes and peripheral neutrophils retain expression making Ly6G a good cell surface marker for these populations. Unlike the RB6-8C5 antibody, the 1A8 antibody reacts specifically with mouse Ly6G with no reported cross reactivity with Ly6C.Specifications
| Isotype | Rat IgG2a,Ā Īŗ |
|---|---|
| Conjugation | APC |
| Excitation Source | Red 627-640 nm |
| Excitation Max | 651 nm |
| Emission Max | 660 nm |
| Immunogen | EL4J cells transfected with Ly6G |
| Reported Applications |
Immunofluorescence Immunohistochemistry (paraffin) Immunohistochemistry (frozen) Flow cytometry |
| Protocol Information | It is recommended that the reagent be carefully titrated for optimal performance in the assay of interest. |
| Concentration | 0.2 mg/ml |
| Formulation |
PBS, pH 7.0 Contains 0.09% Sodium Azide |
| Purification | Protein G |
| RRID | AB_1107721 |
| Storage | The antibody solution should be stored at the stock concentration at 4Ā°C and protected from prolonged exposure to light. Do not freeze. |
Additional Formats
Recommended Products
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Recommended Dilution Buffer
InVivoPure pH 7.0 Dilution Buffer
Flow Cytometry, in vivo neutrophil depletion
Conventional DCs reduce liver ischemia/reperfusion injury in mice via IL-10 secretion PubMed
TLRs are recognized as promoters of tissue damage, even in the absence of pathogens. TLR binding to damage-associated molecular patterns (DAMPs) released by injured host cells unleashes an inflammatory cascade that amplifies tissue destruction. However, whether TLRs possess the reciprocal ability to curtail the extent of sterile inflammation is uncertain. Here, we investigated this possibility in mice by studying the role of conventional DCs (cDCs) in liver ischemia/reperfusion (I/R) injury, a model of sterile inflammation. Targeted depletion of mouse cDCs increased liver injury after I/R, as assessed by serum alanine aminotransferase and histologic analysis. In vitro, we identified hepatocyte DNA as an endogenous ligand to TLR9 that promoted cDCs to secrete IL-10. In vivo, cDC production of IL-10 required TLR9 and reduced liver injury. In addition, we found that inflammatory monocytes recruited to the liver via chemokine receptor 2 were downstream targets of cDC IL-10. IL-10 from cDCs reduced production of TNF, IL-6, and ROS by inflammatory monocytes. Our results implicate inflammatory monocytes as mediators of liver I/R injury and reveal that cDCs respond to DAMPS during sterile inflammation, providing the host with protection from progressive tissue damage.
Immunofluorescence, in vivo neutrophil depletion
CD8alpha(+) dendritic cells are an obligate cellular entry point for productive infection by Listeria monocytogenes PubMed
CD8alpha(+) dendritic cells (DCs) prime cytotoxic T lymphocytes during viral infections and produce interleukin-12 in response to pathogens. Although the loss of CD8alpha(+) DCs in Batf3(-/-) mice increases their susceptibility to several pathogens, we observed that Batf3(-/-) mice exhibited enhanced resistance to the intracellular bacterium Listeria monocytogenes. In wild-type mice, Listeria organisms, initially located in the splenic marginal zone, migrated to the periarteriolar lymphoid sheath (PALS) where they grew exponentially and induced widespread lymphocyte apoptosis. In Batf3(-/-) mice, however, Listeria organisms remain trapped in the marginal zone, failed to traffic into the PALS, and were rapidly cleared by phagocytes. In addition, Batf3(-/-) mice, which lacked the normal population of hepatic CD103(+) peripheral DCs, also showed protection from liver infection. These results suggest that Batf3-dependent CD8alpha(+) and CD103(+) DCs provide initial cellular entry points within the reticuloendothelial system by which Listeria establishes productive infection.
Flow Cytometry, in vivo neutrophil depletion
Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway PubMed
Trehalose 6,6ā²-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFalpha production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle(-)/(-) mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.
Flow Cytometry, in vivo neutrophil depletion
Differential role of the interleukin-17 axis and neutrophils in resolution of inhalational anthrax PubMed
The roles of interleukin-17 (IL-17) and neutrophils in the lung have been described as those of two intricate but independent players. Here we identify neutrophils as the primary IL-17-secreting subset of cells in a model of inhalation anthrax using A/J and C57BL/6 mice. With IL-17 receptor A knockout (IL-17RA-/-) mice, we confirmed that IL-17A/F signaling is instrumental in the self-recruitment of this population. We also show that the IL-17A/F axis is critical for surviving pulmonary infection, as IL-17RA-/- mice become susceptible to intranasal infection by Bacillus anthracis Sterne spores. Strikingly, infection with a fully virulent strain did not affect IL-17RA-/- mouse survival. Eventually, by depleting neutrophils in wild-type and IL-17RA-/- mice, we demonstrated the crucial role of IL-17-secreting neutrophils in mouse survival of infection by fully virulent B. anthracis. This work demonstrates the important roles of both IL-17 signaling and neutrophils in clearing this pathogen and surviving pulmonary B. anthracis infection.
Flow Cytometry, Immunohistochemistry (frozen), in vivo neutrophil depletion
Intrahepatic myeloid-cell aggregates enable local proliferation of CD8(+) T cells and successful immunotherapy against chronic viral liver infection PubMed
Chronic infection is difficult to overcome because of exhaustion or depletion of cytotoxic effector CD8(+) T cells (cytotoxic T lymphoytes (CTLs)). Here we report that signaling via Toll-like receptors (TLRs) induced intrahepatic aggregates of myeloid cells that enabled the population expansion of CTLs (iMATEs: āintrahepatic myeloid-cell aggregates for T cell population expansionā) without causing immunopathology. In the liver, CTL proliferation was restricted to iMATEs that were composed of inflammatory monocyte-derived CD11b(+) cells. Signaling via tumor-necrosis factor (TNF) caused iMATE formation that facilitated costimulation dependent on the receptor OX40 for expansion of the CTL population. The iMATEs arose during acute viral infection but were absent during chronic viral infection, yet they were still induced by TLR signaling. Such hepatic expansion of the CTL population controlled chronic viral infection of the liver after vaccination with DNA. Thus, iMATEs are dynamic structures that overcome regulatory cues that limit the population expansion of CTLs during chronic infection and can be used in new therapeutic vaccination strategies.
Flow Cytometry, in vivo IL-1Ī² neutralization, in vivo neutrophil depletion
The neutrophil NLRC4 inflammasome selectively promotes IL-1beta maturation without pyroptosis during acute Salmonella challenge PubMed
The macrophage NLRC4 inflammasome drives potent innate immune responses against Salmonella by eliciting caspase-1-dependent proinflammatory cytokine production (e.g., interleukin-1beta [IL-1beta]) and pyroptotic cell death. However, the potential contribution of other cell types to inflammasome-mediated host defense against Salmonella was unclear. Here, we demonstrate that neutrophils, typically viewed as cellular targets of IL-1beta, themselves activate the NLRC4 inflammasome during acute Salmonella infection and are a major cell compartment for IL-1beta production during acute peritoneal challenge in vivo. Importantly, unlike macrophages, neutrophils do not undergo pyroptosis upon NLRC4 inflammasome activation. The resistance of neutrophils to pyroptotic death is unique among inflammasome-signaling cells so far described and allows neutrophils to sustain IL-1beta production at a site of infection without compromising the crucial inflammasome-independent antimicrobial effector functions that would be lost if neutrophils rapidly lysed upon caspase-1 activation. Inflammasome pathway modification in neutrophils thus maximizes host proinflammatory and antimicrobial responses during pathogen challenge.
Flow Cytometry, in vivo CD80 blockade, In vivo CD86 blockade, in vivo neutrophil depletion
Late engagement of CD86 after influenza virus clearance promotes recovery in a FoxP3+ regulatory T cell dependent manner PubMed
Influenza A virus (IAV) infection in the respiratory tract triggers robust innate and adaptive immune responses, resulting in both virus clearance and lung inflammation and injury. After virus clearance, resolution of ongoing inflammation and tissue repair occur during a distinct recovery period. B7 family co-stimulatory molecules such as CD80 and CD86 have important roles in modulating T cell activity during the initiation and effector stages of the host response to IAV infection, but their potential role during recovery and resolution of inflammation is unknown. We found that antibody-mediated CD86 blockade in vivo after virus clearance led to a delay in recovery, characterized by increased numbers of lung neutrophils and inflammatory cytokines in airways and lung interstitium, but no change in conventional IAV-specific T cell responses. However, CD86 blockade led to decreased numbers of FoxP3+ regulatory T cells (Tregs), and adoptive transfer of Tregs into alphaCD86 treated mice rescued the effect of the blockade, supporting a role for Tregs in promoting recovery after virus clearance. Specific depletion of Tregs late after infection mimicked the CD86 blockade phenotype, confirming a role for Tregs during recovery after virus clearance. Furthermore, we identified neutrophils as a target of Treg suppression since neutrophil depletion in Treg-depleted mice reduced excess inflammatory cytokines in the airways. These results demonstrate that Tregs, in a CD86 dependent mechanism, contribute to the resolution of disease after IAV infection, in part by suppressing neutrophil-driven cytokine release into the airways.
Flow Cytometry, Immunofluorescence, Immunohistochemistry (frozen), Immunohistochemistry (paraffin), in vivo neutrophil depletion
MET is required for the recruitment of anti-tumoural neutrophils PubMed
Mutations or amplification of the MET proto-oncogene are involved in the pathogenesis of several tumours, which rely on the constitutive engagement of this pathway for their growth and survival. However, MET is expressed not only by cancer cells but also by tumour-associated stromal cells, although its precise role in this compartment is not well characterized. Here we show that MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor (HGF). Met deletion in mouse neutrophils enhances tumour growth and metastasis. This phenotype correlates with reduced neutrophil infiltration to both the primary tumour and metastatic sites. Similarly, Met is necessary for neutrophil transudation during colitis, skin rash or peritonitis. Mechanistically, Met is induced by tumour-derived tumour necrosis factor (TNF)-alpha or other inflammatory stimuli in both mouse and human neutrophils. This induction is instrumental for neutrophil transmigration across an activated endothelium and for inducible nitric oxide synthase production upon HGF stimulation. Consequently, HGF/MET-dependent nitric oxide release by neutrophils promotes cancer cell killing, which abates tumour growth and metastasis. After systemic administration of a MET kinase inhibitor, we prove that the therapeutic benefit of MET targeting in cancer cells is partly countered by the pro-tumoural effect arising from MET blockade in neutrophils. Our work identifies an unprecedented role of MET in neutrophils, suggests a potential āAchillesā heelā of MET-targeted therapies in cancer, and supports the rationale for evaluating anti-MET drugs in certain inflammatory diseases.
Flow Cytometry, Immunohistochemistry (paraffin), in vivo CD8+ T cell depletion, in vivo IL-17A neutralization, in vivo IL-1Ī² neutralization, in vivo IL-23p19 neutralization, in vivo neutrophil depletion
IL-17-producing gammadelta T cells and neutrophils conspire to promote breast cancer metastasis PubMed
Metastatic disease remains the primary cause of death for patients with breast cancer. The different steps of the metastatic cascade rely on reciprocal interactions between cancer cells and their microenvironment. Within this local microenvironment and in distant organs, immune cells and their mediators are known to facilitate metastasis formation. However, the precise contribution of tumour-induced systemic inflammation to metastasis and the mechanisms regulating systemic inflammation are poorly understood. Here we show that tumours maximize their chance of metastasizing by evoking a systemic inflammatory cascade in mouse models of spontaneous breast cancer metastasis. We mechanistically demonstrate that interleukin (IL)-1beta elicits IL-17 expression from gamma delta (gammadelta) T cells, resulting in systemic, granulocyte colony-stimulating factor (G-CSF)-dependent expansion and polarization of neutrophils in mice bearing mammary tumours. Tumour-induced neutrophils acquire the ability to suppress cytotoxic T lymphocytes carrying the CD8 antigen, which limit the establishment of metastases. Neutralization of IL-17 or G-CSF and absence of gammadelta T cells prevents neutrophil accumulation and downregulates the T-cell-suppressive phenotype of neutrophils. Moreover, the absence of gammadelta T cells or neutrophils profoundly reduces pulmonary and lymph node metastases without influencing primary tumour progression. Our data indicate that targeting this novel cancer-cell-initiated domino effect within the immune systemāthe gammadelta T cell/IL-17/neutrophil axisārepresents a new strategy to inhibit metastatic disease.