Ly-6G/Ly-6C Antibody (RB6-8C5) - BSA Free

Western Blot: Ly-6G/Ly-6C Antibody (RB6-8C5) [NBP2-00441] - Mice were treated with oil in combination with injection of lenti-NC (NC, n = 10), CCl4 in combination with injection of lenti-NC (NC + CCl4, n = 10), oil in combination with injection of lenti-lnc-Lfar1-shRNA (lnc-Lfar1-shRNA, n = 10), and CCl4 in combination with injection of lenti-lnc-Lfar1-shRNA (lnc-Lfar1-shRNA + CCl4, n = 10). b The protein level of F4/80, CD11b and LY6C was determined by western blot. GAPDH was used as an internal control. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41419-020-2323-5), licensed under a CC-BY license.

Immunohistochemistry: Ly-6G/Ly-6C Antibody (RB6-8C5) [NBP2-00441] - Mice were treated with oil in combination with injection of lenti-NC (NC, n = 10), CCl4 in combination with injection of lenti-NC (NC + CCl4, n = 10), oil in combination with injection of lenti-lnc-Lfar1-shRNA (lnc-Lfar1-shRNA, n = 10), and CCl4 in combination with injection of lenti-lnc-Lfar1-shRNA (lnc-Lfar1-shRNA + CCl4, n = 10). a Immunohistochemistry analysis was performed to detect the expression of F4/80 and LY6C; scale bar = 400 um for 10 x and 100 um for 40x. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41419-020-2323-5), licensed under a CC-BY license.

Flow Cytometry: Ly-6G/Ly-6C Antibody (RB6-8C5) [NBP2-00441] - 1 ug/1^6 BALB/c bone marrow cells were stained with either rat IgG2b: FITC (as an isotype control) or rat anti-mouse Ly-6G: FITC. Large cells were then gated and analyzed on a flow cytometer.

Flow Cytometry: Ly-6G/Ly-6C Antibody (RB6-8C5) [NBP2-00441] - Analysis using the Allophycocyanin conjugate of NBP2-00441. Staining of C57BL/6 bone marrow cells with 0.125 ug of Rat IgG2b kappa Isotype Control APC (blue histogram) or 0.125 ug of Anti-Mouse Ly-6G (Gr-1) APC (purple histogram).

Immunohistochemistry-Paraffin: Ly-6G/Ly-6C Antibody (RB6-8C5) [NBP2-00441] - Analysis of a FFPE tissue section of mouse bone marrow using 1:100 dilution of Lot E04586-1632 of Ly-6G antibody (clone RB6-8C5). The antibody generated specific staining in a subset of cells in the tested section.

Immunohistochemistry-Paraffin: Ly-6G/Ly-6C Antibody (RB6-8C5) [NBP2-00441] - Analysis of a FFPE tissue section of mouse bone marrow using 1:200 dilution of Lot A-1 of Ly-6G antibody (clone RB6-8C5). The antibody generated specific staining in a subset of cells in the tested section. The neutrophils (identifiable from typical nuclear morphology) showed stronger signal than the neighboring cells.

Flow Cytometry: Ly-6G/Ly-6C Antibody (RB6-8C5) [NBP2-00441] - Staining of mouse bone marrow with Anti-Mouse Ly-6G (Gr-1) FITC and Anti-Human/Mouse CD45R (B220) PE. Total viable cells were used for analysis.

ICM and TE progenitors show loss of responsiveness to Hippo signaling manipulation at the same time as they loose responsiveness to positional changes.(A) Overview of Hippo signaling activation time course. Each bar represents 24 hr of 50 µM ROCKi treatment. (B) Percent of Cdx2 positive cells per embryo cultured for 24 hr in control or ROCKi conditions. Label on top indicates the stage embryos started treatment. n indicates number of embryos analyzed. Statistical significance was calculated using t-test and significant p-values are indicated. Error bars: s.d. of mean. (C) Strategy for inducible Hippo signaling inactivation. Mostly mosaic Dox-inducible DN Lats2-IRES-mCherry transgenic embryos were generated. Each bar represents 24 hr of Dox treatment. (D) Dox-inducible DN Lats2-IRES-mCherry transgenic embryos were imaged before Dox treatment (top panel) and the same embryo was imaged following 24 hr of Dox live (middle panel) and fixed/stained for lineage markers (bottom panel). A representative embryo is shown for each stage. Live mCherry is shown as an extended focus image, immunofluorescence stainings shown as single plane images. mCherry positive ICMs in mosaic transgenic embryos are circled with a dotted line. Arrow points to a rare ICM cell in a 64 cell stage-induced embryo with weak Cdx2 expression, which also co-expressed an ICM marker. Scale bar: 25 µm. n indicates number of transgenic embryos analyzed. (E) All mCherry negative (non-transgenic control) and mCherry positive (DN Lats2-mCherry transgenic) ICM cells were scored in mosaic embryos for presence or absence of lineage markers following 24 hr of Dox treatment by immunofluorescence staining. Cells with different lineage marker expression are shown as percent of all mCherry negative or mCherry positive ICM cells analyzed. n(cell) indicates number of cells analyzed at each stage and n(embryo) indicates number of embryos cells were pooled from. Chi-squared test was used to test whether cell fate was affected by DN Lats2-mCherry expression. 16 cell p-value=8.48491E-18; early 32 cell p-value=5.50841E-34; late 32 cell p-value=6.32116E-35; 64 cell p-value=0.004103716; >64 cell p-value=0.588416983.DOI://dx.doi.org/10.7554/eLife.22906.019Effect of ROCKi treatment on cell number and Hippo signaling.(A) Total cell numbers in control and 50 µM ROCKi treated embryos at different stages. n indicates number of embryos analyzed. Statistical significance was calculated using t-test and significant p-values are indicated. Error bars: s.d. of mean. (B) Immunofourescence staining of control and 50 µM ROCKi treated embryos for TE marker (Cdx2), ICM marker (Klf4) and Yap. 24 hr treatment was started at the 16 cell stage. A total of 4 control and 4 ROCKi-treated embryos were imaged in one experiment. Scale bar: 25 µm. (C) Immunofourescence staining of control and 50 µM ROCKi treated embryos for TE marker (Cdx2), ICM marker (Klf4) and phospho-Yap (form of Yap sequestered into the cytoplasm due to active Hippo signaling). 24 hr treatment was started at the 16 cell stage. A total of 4 control and 3 ROCKi-treated embryos were imaged in one experiment. Scale bar: 25 µm.DOI://dx.doi.org/10.7554/eLife.22906.020Expression of mCherry only does not influence cell fate in the embryo.2 cell stage embryos were injected with H2O (wild-type control) or a cocktail of PB-TAC-mCherry-IRES-mCherry, PB-CAG-rtTA and PBase mRNA (mCherry control). Embryos were treated with Dox for 24 hours starting at the 32 or 64 cell stages. Following Dox treatment cell fate of ICM cells was analyzed by immunofluorescence staining for lineage markers. Cell fates shown as percent of all H2O injected ICM cells (in H2O injected embryos) or all mCherry positive ICM cells (in mCherry control embryos). n(cell) indicates number of cells analyzed at each stage and n(embryo) indicates number of embryos cells were pooled from. Chi-squared test was used to test whether cell fate was affected by mCherry expression. 32 cell p-value= 0.139370244, 64 cell p-value= 0.07551351.DOI://dx.doi.org/10.7554/eLife.22906.021 Image collected and cropped by CiteAb from the following open publication (//elifesciences.org/articles/22906), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: Ly-6G/Ly-6C Antibody (RB6-8C5) - BSA Free [NBP2-00441] - Silencing lnc-Lfar1 alleviates CCl4-induced proinflammatory activation of macrophages.Mice were treated with oil in combination with injection of lenti-NC (NC, n = 10), CCl4 in combination with injection of lenti-NC (NC + CCl4, n = 10), oil in combination with injection of lenti-lnc-Lfar1-shRNA (lnc-Lfar1-shRNA, n = 10), & CCl4 in combination with injection of lenti-lnc-Lfar1-shRNA (lnc-Lfar1-shRNA + CCl4, n = 10). a Immunohistochemistry analysis was performed to detect the expression of F4/80 & LY6C; scale bar = 400 μm for 10 × and 100 μm for 40×. b The protein level of F4/80, CD11b & LY6C was determined by western blot. GAPDH was used as an internal control. c The mRNA level of F4/80, Ly6c, Ccr2, Cd20, Il-6, iNos, Ccl5, Cxcl5, Cxcl9 & Cxcl10 was determined by qRT-PCR. *p < 0.05 vs NC, #p < 0.05 vs NC + CCl4. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32071306), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

• Reactivity: Hu, Mu
• Applications: WB, Flow, Func, ICC/IF, IHC, IP, CyTOF-ready
• Clone: RB6-8C5
• Clonality: Monoclonal
• Host: Rat
• Conjugate: Unconjugated
• Format: BSA Free
• Concentration: 1.0 mg/ml

Ly-6G/Ly-6C Antibody (RB6-8C5) - BSA Free Summary

• Description: Novus Biologicals Rat Ly-6G/Ly-6C Antibody (RB6-8C5) - BSA Free (NBP2-00441) is a monoclonal antibody validated for use in IHC, WB, Flow, ICC/IF and IP. Anti-Ly-6G/Ly-6C Antibody: Cited in 25 publications. All Novus Biologicals antibodies are covered by our 100% guarantee.
• Immunogen: Normal mouse bone marrow.
• Specificity: Studies indicate that in the bone marrow and lysed whole blood, the antibody clone RB6-8C5 also reacts with an additional subpopulation of Ly-6C high cells.
• Isotype: IgG2b Kappa
• Clonality: Monoclonal
• Host: Rat
• Gene: Ly6g
• Purity: Protein A or G purified

Applications/Dilutions

• Dilutions:
  • CyTOF-ready
  • Flow Cytometry 1:10-1:1000
  • Immunocytochemistry/ Immunofluorescence 8-25 ug/ml
  • Immunohistochemistry 1:10-1:500
  • Immunohistochemistry-Frozen 1:10-1:500
  • Immunohistochemistry-Paraffin 1:10-1:500
  • Immunoprecipitation 1:10-1:500
  • In vivo assay reported in scientific literature (PMID 16272176)
  • Western Blot 1:100-1:2000
• Application Notes: The RB6-8C5 antibody has been tested by flow cytometric analysis of mouse bone marrow cells and splenocyte suspensions. This can be used at less than or equal to 0.5 ug per test. A test is defined as the amount (ug) of antibody that will stain a cell sample in a final volume of 100 ul. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. The RB6-8C5 antibody has also been reported for use immunoprecipitation, immunoblotting (WB) and immunohistochemical staining. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. This antibody is CyTOF ready.

Reactivity Notes

Use in Mouse reported in scientific literature (PMID:34449927).

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: PBS
• Preservative: 0.02% Sodium Azide
• Concentration: 1.0 mg/ml
• Purity: Protein A or G purified

Alternate Names for Ly-6G/Ly-6C Antibody (RB6-8C5) - BSA Free

• GR
• Gr1
• Gr-1
• Ly-6
• Ly6G
• Ly-6G

Background

The lymphocyte antigen 6 (Ly-6) complex was first described in mice and since its discovery over 20 genes related to the Ly-6 complex on chromosome 15 have been classified (1). The Ly-6 family is commonly used as a cell-surface differentiation marker on both normal and abnormal cells (2). Ly-6 complex, locus G (Ly-6G) and Ly-6 complex, locus C (Ly-6C) both belong to the murine Ly6/urokinase-type plasminogen activator receptor (uPAR) family of genes, which share a common structure (3). The Ly-6 family of proteins is characterized by a ~90 amino acid (aa) LU domain that consists of a three-finger structure formed by disulfide bonds (1,3). Additionally, many Ly6 proteins have a C-terminal GPI anchor, including Ly-6G/Ly-6C (1,3). Ly-6C protein has a molecular weight of 14 kDa and is expressed in some CD4+ and CD8+ T-cells, monocytes, neutrophils, and dendritic cells (1). Ly-6C has uniform expression in neutrophils but varies in monocytes where Ly-6Chi expression is specific for immature/inflammatory monocytes and Ly-6Clo expression identifies mature/residential monocytes (1,3). Ly-6G, on the other hand, is a protein with a molecular weight of 25 kDa with its expression uniquely restricted to neutrophils (1).

Expression of Ly-6G and Ly-6C, along with other lineage markers, help define specific cell types (2,4). For example, mouse myeloid derived suppressor cells (MDSCs), which play a role in inflammation and tumor development, are further subdivided into granulocytic (G-MDSCs) or monocytic (Mo-MDSCs) (4). Mo-MDSCs express Ly-6Chi along with CD11b, but are negative for Ly-6G, whereas G-MDSCs express Ly-6Clo/int,Ly-6Ghi , and CD11b (4).

The monoclonal antibody RB6-8C5 first identified granulocyte-differentiation antigen (Gr-1) which is primarily expressed on the surface of neutrophils and immature myeloid cells, and to a lesser extent on lymphocytes and macrophages (1). Gr-1 expression increases as neutrophils mature but remains is stably expressed in monocytes (1). The RB6-8C5 reacts with both Ly-6G and Ly-6C, whereas 1A8 antibody is specific for Ly-6G (1). Antibodies against Ly-6G, such as RB6-8C5 and 1A8, are commonly used to identify a role for neutrophils in mouse models of disease as they are efficient for quickly depleting neutrophils (1). RB6-8C5, however, also targets and depletes Ly-6Chi monocytes in addition to other Ly-6G-expressing cells (1).

References

1. Lee, P. Y., Wang, J. X., Parisini, E., Dascher, C. C., & Nigrovic, P. A. (2013). Ly6 family proteins in neutrophil biology. Journal of leukocyte biology. https://doi.org/10.1189/jlb.0113014

2. Bamezai A. (2004). Mouse Ly-6 proteins and their extended family: markers of cell differentiation and regulators of cell signaling. Archivum immunologiae et therapiae experimentalis.

3. Loughner, C. L., Bruford, E. A., McAndrews, M. S., Delp, E. E., Swamynathan, S., & Swamynathan, S. K. (2016). Organization, evolution and functions of the human and mouse Ly6/uPAR family genes. Human genomics. https://doi.org/10.1186/s40246-016-0074-2

4. Kong, Y. Y., Fuchsberger, M., Xiang, S. D., Apostolopoulos, V., & Plebanski, M. (2013). Myeloid derived suppressor cells and their role in diseases. Current medicinal chemistry. https://doi.org/10.2174/0929867311320110006

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for Ly-6G/Ly-6C Antibody (NBP2-00441)(25)

Publications using NBP2-00441

• Zhang K, Zhang MX, Meng XX et al. Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-?B pathways Military Medical Research 2023-11-25 [PMID: 38001521] (IHC, WB, Human, Mouse)
• Tang N, Gong XR, Huang H, Meng Q Activated neutrophil-derived exosomes contribute to blood-brain barrier damage and hemorrhagic transformation after cerebral ischemia/reperfusion Brain research 2023-04-26 [PMID: 37116559] (IHC, Mouse)
• Li J, Zou X, Yang S Et al. Neutrophil Extracellular Traps Participate in the Development of Gastric Cancer Associated Thrombosis Research Square 2021-07-27 [PMID: 36051331] (ICC/IF)
• Ruhela D, Bhopale VM, Kalakonda S et al. Astrocyte-derived microparticles initiate a neuroinflammatory cycle due to carbon monoxide poisoning Brain Behav Immun Health 2021-12-01 [PMID: 34917988] (WB, FLOW, Mouse)
  
  Details:
  Citation using the PerCP format of this antibody.
• Spencer NR, Radnaa E, Baljinnyam T Et al. Development of a mouse model of ascending infection and preterm birth PLoS One 2021-12-02 [PMID: 34855804] (IHC-P, Mouse)
  
  Details:
  Citation using the DyLight 650 version of this antibody.
• Zhang S, Cao Y, Du J Et al. Neutrophil extracellular traps contribute to tissue plasminogen activator resistance in acute ischemic stroke FASEB journal : official publication of the Federation of American Societies for Experimental Biology Sep 1 2021 [PMID: 34449927] (ICC/IF, Human)
• Keshava S, Magisetty J, Tucker TA et al. Endothelial Cell Protein C Receptor Deficiency Attenuates Streptococcus Pneumoniae-induced Pleural Fibrosis American journal of respiratory cell and molecular biology 2021-02-18 [PMID: 33600743]
• Kondreddy V, Pendurthi Ur, Xu X et Al. FVIIa (Factor VIIa) Induces Biased Cytoprotective Signal in Mice Through the Cleavage of PAR (Protease-Activated Receptor)-1 at Canonical Arg41 Site Arterioscler. Thromb. Vasc. Biol. 2020-03-26 [PMID: 32212848] (Mouse)
• Zhang K, Shi Z et al. Silencing lncRNA Lfar1 alleviates the classical activation and pyoptosis of macrophage in hepatic fibrosis. Cell Death Dis 2020-02-18 [PMID: 32071306] (WB, IF/IHC, Mouse)
• Miyabe C, Miyabe Y, Moreno L et al. Dectin-2-induced CCL2 production in tissue-resident macrophages ignites cardiac arteritis J. Clin. Invest. 2019-06-06 [PMID: 31169521] (IHC-P, Mouse)
• Hathaway-Schrader JD, Steinkamp HM, Chavez MB et al. Antibiotic Perturbation of Gut Microbiota Dysregulates Osteoimmune Cross Talk in Postpubertal Skeletal Development. Am. J. Pathol. 2019-02-01 [PMID: 30660331] (FLOW, Mouse)
• Kishimoto A, Kimura S, Nio-Kobayashi J et al. Histochemical characteristics of regressing vessels in the hyaloid vascular system of neonatal mice: Novel implication for vascular atrophy. Exp. Eye Res. 2018-03-27 [PMID: 29596849] (Mouse)
• Benedicto A, Romayor I, Arteta B. CXCR4 receptor blockage reduces the contribution of tumor and stromal cells to the metastatic growth in the liver. Oncol Rep. 2018-02-08 [PMID: 29436696] (Mouse)
  
  Details:
  This citation used the Biotin form of this antbody
• Murase T, Yamamoto T, Koide A et al. Temporal expression of chitinase-like 3 in wounded murine skin Int. J. Legal Med. 2017-08-06 [PMID: 28780759] (IF/IHC, Mouse)
• Stremnitzer C, Manzano-Szalai K et al. Papain Degrades Tight Junction Proteins of Human Keratinocytes In Vitro and Sensitizes C57BL/6 Mice via the Skin Independent of its Enzymatic Activity or TLR4 Activation. J Invest Dermatol 2015-01-07 [PMID: 25705851] (IF/IHC, Mouse)
• Pickens SR, Chamberlain ND, Volin MV et al. Local expression of interleukin-27 ameliorates collagen-induced arthritis Arthritis Rheum 2011-08-01 [PMID: 21384333] (IF/IHC, Mouse)
• Huang B, Zhao J, Shen S, Li H, He KL, Shen GX, Mayer L, Unkeless J, Li D, Yuan Y, Zhang GM, Xiong H, Feng ZH. Listeria monocytogenes promotes tumor growth via tumor cell toll-like receptor 2 signaling. Cancer Res;67(9):4346-52. 2007-05-01 [PMID: 17483348] (In Vivo)
• Sasmono RT, Ehrnsperger A, Cronau SL, Ravasi T, Kandane R, Hickey MJ, Cook AD, Himes SR, Hamilton JA, Hume DA. Mouse neutrophilic granulocytes express mRNA encoding the macrophage colony-stimulating factor receptor (CSF-1R) as well as many other macrophage-specific transcripts and can transdifferentiate into macrophages in vitro in response to CSF-1. J Leukoc Biol;82(1):111-23. 2007-07-01 [PMID: 17438263]
• Fan J, Li Y, Vodovotz Y, Billiar TR, Wilson MA. Hemorrhagic shock-activated neutrophils augment TLR4 signaling-induced TLR2 upregulation in alveolar macrophages: role in hemorrhage-primed lung inflammation. Am J Physiol Lung Cell Mol Physiol;290(4):L738-L746. 2006-04-01 [PMID: 16272176] (In Vivo)
• Alvarez MJ, Prada F, Salvatierra E, Bravo AI, Lutzky VP, Carbone C, Pitossi FJ, Chuluyan HE, Podhajcer OL. Secreted protein acidic and rich in cysteine produced by human melanoma cells modulates polymorphonuclear leukocyte recruitment and antitumor cytotoxic capacity. Cancer Res;65(12):5123-32. 2005-06-15 [PMID: 15958556]
• Brown CR, Blaho VA, Loiacono CM. Susceptibility to experimental Lyme arthritis correlates with KC and monocyte chemoattractant protein-1 production in joints and requires neutrophil recruitment via CXCR2. J Immunol;171(2):893-901. 2003-07-15 [PMID: 12847259] (IHC-P)
• McLellan AD, Kapp M, Eggert A et al. Anatomic location and T-cell stimulatory functions of mouse dendritic cell subsets defined by CD4 and CD8 expression. Blood. 2002-03-15 [PMID: 11877283] (FLOW, Mouse)
  
  Details:
  This citation used the FITC version of this antibody.
• Miura M, Fu X, Zhang QW, Remick DG, Fairchild RL. Neutralization of Gro alpha and macrophage inflammatory protein-2 attenuates renal ischemia/reperfusion injury. Am J Pathol;159(6):2137-45. 2001-12-01 [PMID: 11733364] (IHC-Fr)
• Dunussi-Joannopoulos K, Runyon K, Erickson J, Schaub RG, Hawley RG, Leonard JP. Vaccines with interleukin-12-transduced acute myeloid leukemia cells elicit very potent therapeutic and long-lasting protective immunity. Blood;94(12):4263-73. 1999-12-15 [PMID: 10590071] (IHC-Fr)
• Fleming TJ, Fleming ML, Malek TR. Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocytedifferentiation antigen (Gr-1) detects members of the Ly-6 family. J Immunol 1993-09-01 [PMID: 8360469]

Reviews for Ly-6G/Ly-6C Antibody (NBP2-00441) (2)

Average Rating: 4.5

(Based on 2 reviews)

We have 2 reviews tested in 1 species: Mouse.

reviewed by: Verified Customer IHC-P Mouse 02/09/2021

Summary

• Application: Immunohistochemistry-Paraffin
• Sample Tested: Fetal membranes
• Species: Mouse
• Lot: A-6

reviewed by: Verified Customer IHC-P Mouse 02/24/2017

Summary

• Application: Immunohistochemistry-Paraffin
• Sample Tested: mouse skin
• Species: Mouse

Comments

• Comments: dilution 1:100
  Overnight incubation

FAQs for Ly-6G/Ly-6C Antibody (NBP2-00441). (Showing 1 - 2 of 2 FAQs).

1. On the Datasheet of Ly-6G Antibody (RB6-8C5), Western Blot is listed as one of the applications. Do you have any suggestions on the concentration to use for this Ab in WB?
   • We recommend dilutions between 1:1000 and 1:2000 as a starting point in Western blot.
2. Can you please provide me with any publication reference the uses clone RB6-8C5 in Western?
   • Unfortunately we do not have any references for use with this antibody in Western blot at the time.

Bioinformatics

• Gene Symbol: Ly6g