Ly6g 1a8

Manufactured by BD

Sourced in United States

Ly6G (1A8) is a monoclonal antibody that recognizes the Ly-6G antigen, which is expressed on the surface of neutrophils. It can be used for the identification and enumeration of neutrophils in various applications.

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Lab products found in correlation

Cd11b m1 70, by Thermo Fisher Scientific (8 mentions) Cd11b m1 70, by BioLegend (8 mentions) Ly6c hk1, by Thermo Fisher Scientific (7 mentions) Cd11b m1 70, by BD (7 mentions) Cd45 30 f11, by Thermo Fisher Scientific (6 mentions) Ly6c al 21, by BD (5 mentions) Foxp3 fjk 16s, by Thermo Fisher Scientific (5 mentions) Cd206 c068c2, by BioLegend (4 mentions) Cd45 30 f11, by BD (4 mentions) B220 ra3 6b2, by BioLegend (4 mentions) Facscanto 2, by BD (4 mentions) Ly6c hk1, by BioLegend (4 mentions) Cd11c n418, by BioLegend (3 mentions) Cd8 53 6, by Thermo Fisher Scientific (3 mentions) Cd68 fa 11, by Bio-Rad (3 mentions) Facsaria, by BD (3 mentions) Cd64 x54 5 7, by BioLegend (3 mentions) Siglecf e50 2440, by BD (3 mentions) F4 80 bm8, by BioLegend (3 mentions) Dnase 1, by Roche (3 mentions)

Spelling variants of this product

Anti-Ly6G (clone 1A8, (17 citations) FITC-conjugated anti-Ly6G (clone 1A8, (8 citations) Anti-Ly6G-PE (clone 1A8) (6 citations) Ly6G‐PE (1A8; (3 citations) Anti-mouse Ly6G (1A8, (3 citations) Anti-Ly6G-APC-Cy7 (1A8, (3 citations) Rat monoclonal antibody to Ly6G ([Gr-1] clone 1A8; (2 citations) Ly6G 1A8-PE Cy7 (2 citations) PE) conjugated anti-Ly6G (clone 1A8, (2 citations) CD11b-PE and Ly6G-FITC clone 1A8 (2 citations)

44 protocols using ly6g 1a8

Multiparametric Flow Cytometry of Immune Cells

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Single cell suspensions were incubated with an anti-FcγIII/II receptor antibody (clone 93) to block unspecific binding and Zombie Violet^TM (Biolegend, Germany), a fixable viability dye. Thereafter, cells were stained with fluorochrome conjugated antibodies against cell surface markers: CD45 (30-F11, eBioscience, Germany), CD11b (M1/70, eBioscience, Germany), Ly6G (1A8, BD Biosciences, Germany), Ly6C (HK1.4, eBioscience, Germany), CCR2 (475301, R&D, USA), TREM2 (237920, R&D, USA), CD36 (HM36, Biolegend, Germany), in FACS buffer (PBS containing 2 % FCS and 0.1 % NaN₃) for 30 min on ice and then washed and fixed in 4 % paraformaldehyde (PFA) for 10 min.

Möhle L., Israel N., Paarmann K., Krohn M., Pietkiewicz S., Müller A., Lavrik I.N., Buguliskis J.S., Schott B.H., Schlüter D., Gundelfinger E.D., Montag D., Seifert U., Pahnke J, & Dunay I.R. (2016). Chronic Toxoplasma gondii infection enhances β-amyloid phagocytosis and clearance by recruited monocytes. Acta Neuropathologica Communications, 4, 25.

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Quantifying Peritoneal Macrophage Subsets

Cited 1 time

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Two methods were used to quantify peritoneal macrophages. Total peritoneal cells were counted in a hemacytometer or using an automated cell counter. Then this number was multiplied by the percent of macrophages stained for CD115 (AFS98; eBioscience), ICAM-2 (3C4; BioLegend) and F4/80 (BM8, eBioscience) by flow cytometry. These antibodies allow us to distinguish the minor and major peritoneal macrophage populations (Gautier et al., 2012 (link)), with only the major macrophage population expressing ICAM-2 (Gautier et al., 2012 (link)). Alternatively, peritoneal cells obtained by lavage were incubated with FITC-conjugated anti–ICAM-2 mAb, and then a manual count of the fraction of ICAM-2⁺ cells was made and multiplied by the total number of peritoneal cells. Both methods yielded similar results. Other reagents and mAbs used in flow cytometry were Annexin V (Miltenyi Biotec) and antibodies against Ki67 (B56; BD), active caspase 3 (C92-605; BD), pH3 (D2C8; Cell Signaling technology), TIMD4 (RMT4-54; eBioscience), CD206 (MR5D3; Serotec), LYVE-1 (Abcam), Siglec F (E50-2440; BD), CD11b (M1/70; eBioscience), CD5 (53–7.3; BD), B220 (RA3-6B2; eBioscience), ly 6C/G (RB6-8C5; eBioscience), Ly6-G (1A8; BD), TCRb (H57-597; eBioscience), MHC-II (M5/114.15.2; eBioscience), and MARCO (ED31; Serotec).

Gautier E.L., Ivanov S., Williams J.W., Huang S.C., Marcelin G., Fairfax K., Wang P.L., Francis J.S., Leone P., Wilson D.B., Artyomov M.N., Pearce E.J, & Randolph G.J. (2014). Gata6 regulates aspartoacylase expression in resident peritoneal macrophages and controls their survival. The Journal of Experimental Medicine, 211(8), 1525-1531.

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Murine Immune Cell Profiling by Flow Cytometry

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Cells were released from culture plates using TrypLE and were stained after Fc-block (Invitrogen) with a panel of directly fluorochrome-conjugated mAbs against the following murine molecules (clone; source): Axl (R&D; Minneapolis, MN); CD4 (L3T4; eBioscience; San Diego, CA); CD8 (CD8b.2; Biolegend; San Diego, CA); CD11b (M1/70; eBioscience); CD11c (N418; eBioscience); CD19 (MCA1439F; AB Serotec); CD44 (IM7; eBioscience); CD45 (30-F11; BD); CD80 (16-10A1; BioLegend); CD206 (C068C2; BioLegend); Ly6C (AL-21; BD); Ly6G (1A8; BD, Franklin Lakes, NJ); Mertk (R&D). Experiments were performed on an LSR II flow cytometer and data were analyzed as previously described (25 (link)).

McCubbrey A.L., Nelson J.D., Stolberg V.R., Blakely P.K., McCloskey L., Janssen W.J., Freeman C.M, & Curtis J.L. (2015). miR-34a negatively regulates efferocytosis by tissue macrophages in part via SIRT1. Journal of immunology (Baltimore, Md. : 1950), 196(3), 1366-1375.

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Isolation and Staining of Skin-Derived Cells

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Epidermal and dermal layers of ears were separated after 30 min incubation with 2.4 U/ml Dispase (GIBCO, Carlsbad, CA) at 37°C, followed by cutting into fine pieces and digested with 50 U/ml collagenase for 45 min at 37°C. Single-cell suspension was made in FACS buffer (PBS, 0.5% BSA; Sigma-Aldrich) and blocked with Fc block (eBioscience) and stained with the following antibodies in FACS buffer at 4°C as described before (Chopin et al., 2013 (link)). Antibodies raised against CD11c (N418), CD45.2 (A20), MHCII (M514.15.2), CD8α (53–6.7), CD4 (GK1.4), IFNγ, Ly6G (1A8), were purchased from BD biosciences. TCRβ (H57-597) and cd11b (M1/70) were purchased from eBioscience.

Etemadi N., Chopin M., Anderton H., Tanzer M.C., Rickard J.A., Abeysekera W., Hall C., Spall S.K., Wang B., Xiong Y., Hla T., Pitson S.M., Bonder C.S., Wong W.W., Ernst M., Smyth G.K., Vaux D.L., Nutt S.L., Nachbur U, & Silke J. (2015). TRAF2 regulates TNF and NF-κB signalling to suppress apoptosis and skin inflammation independently of Sphingosine kinase 1. eLife, 4, e10592.

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Comprehensive Immune Cell Profiling

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Lung cells were isolated by collagenase and DNase digestion (Sigma Aldrich). Splenocytes were mashed through 100 µm nylon cell strainers and erythrocytes were lysed in ACK buffer. Lung cells were washed with Hank’s Balanced Salt Solution (HBSS) and splenocytes were washed with PBS. Lung cells and splenocytes were pre-incubated with the 2.4 G2 antibody to block Fc receptor binding, followed by incubation with various cell surface Abs: CD3 (17-A2), NK1.1 (PK136), B220 (RA3-6B2), F4/80 (BM8), MHCII (M5/114.15.2), (eBioscience, San Diego, CA), Siglec F (E50-2440), CD11c (N418), CD11b (M1/70), Gr-1 (RB6-8C5) and Ly6G (1A8) (BD Biosciences, San Jose, CA). Cells were fixed with BD Cytofix and analysed on BD LSRII.

Burg A.R., Quigley L., Jones A.V., O’Connor G.M., Boelte K., McVicar D.W, & Orr S.J. (2016). Orally administered β-glucan attenuates the Th2 response in a model of airway hypersensitivity. SpringerPlus, 5(1), 815.

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Flow Cytometry of Immune Cell Subsets

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Cells were resuspended in staining buffer (PBS with 2% FCS; 5 mM EDTA), blocked with anti-CD16/32 Fc block (BD, Biosciences, Franklin Lakes, NJ, USA), and labeled with fluorochrome-conjugated antibodies for 1 h. Antibodies against CD45 (HI30; BD, Biosciences), CD3 (145-2C11; Invitrogen), CD4 (RM4-5; BioLegend), CD8 (53-6.7; BioLegend), Ly6C (HK1.4; BioLegend), Ly6G (1A8; BD, Bioscience), CD11b (M1/70; BioLegend) and NK1.1 (PK136; BD, Bioscience) were used, and a near infrared (NIR) LIVE/DEAD stain (Life Technology) was used to exclude dead cells. Counting beads (Spherobeads, BD) were added to samples before acquisition. Samples were acquired on a BD LSRFortessa flow cytometer, and data analysis was performed using FlowJo 10.7.

Ng W.H., Liu X., Ling Z.L., Santos C.N., Magalhães L.S., Kueh A.J., Herold M.J., Taylor A., Freitas J.R., Koit S., Wang S., Lloyd A.R., Teixeira M.M., Merits A., Almeida R.P., King N.J, & Mahalingam S. (2023). FHL1 promotes chikungunya and o’nyong-nyong virus infection and pathogenesis with implications for alphavirus vaccine design. Nature Communications, 14, 6605.

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Multiparameter Flow Cytometry Analysis

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Single-cell suspensions from spleens were stained with the Live/Dead Cell Viability assay (Invitrogen), and cell surface markers were stained with the following anti-mouse antibodies at 1:200 dilution per 10⁶ cells: Ly6C (AL-21; BD Biosciences), Ly6G (1A8; BD Biosciences), B220 (RA3-6B2; BioLegend), CD11B (M1/70; BioLegend), TCRb (H57-597; BioLegend), CD11C (N418; BioLegend), and CD45 (30.F11; eBioscience). All samples were analyzed using a FACS Fortessa cytometer (BD Biosciences).

Merritt C., Chun E.M., Fattah R.J., Silva L.M., Ma Q.Q., Moayeri M., Paliga D., Neumann S., Heumann R., Leppla S.H, & Bugge T.H. (2021). Imaging of anthrax intoxication in mice reveals shared and individual functions of surface receptors CMG-2 and TEM-8 in cellular toxin entry. The Journal of Biological Chemistry, 298(1), 101467.

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Multicolor Flow Cytometry for Immune Cell Profiling

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Cells were stained using fluorescently-conjugated antibodies specific for the following molecules: CD64 (X-54-517.1), purchased from Biolegend; Ly6C (AL-21) and Ly6G (1A8), purchased from BD Biosciences; F4/80 (BM8), CD11c (HL3), CD11b (M1/70), CD19 (D3), B220 (RA3-6B2), CD3 (17A2), and streptavidin, purchased from eBioscience. Biotin-conjugated IFNGR1/CD119 (2E2; Biolegend) was detected with PE Cy5-conjugated streptavidin (eBioscience) Dendritic cells were defined as CD11c^hiF4/80^−/lo, macrophages as CD11c^−/lo and F4/80^hi or CD64^hi; B cells as CD3⁻B220⁺CD19⁺; T cells as CD3⁺CD19⁻ or, for Fig. 3, B220⁻CD3⁺TCRβ⁺; and neutrophils as Ly6G^hiLy6C^intCD11b⁺. Fluorescence was measured using a LSRII Flow Cytometer (BD) and analysis was performed using FlowJo V.10 (Treestar).

Pitts M.G., Myers-Morales T, & D’Orazio S.E. (2016). Type I interferon does not promote susceptibility to foodborne Listeria monocytogenes. Journal of immunology (Baltimore, Md. : 1950), 196(7), 3109-3116.

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Kidney Leukocyte Isolation and Neutrophil Depletion

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Kidneys were minced into ~1 mm³ pieces and then digested with 0.5 mg/mL Collagenase D (Sigma), 0.5 mg/mL DNAse (Sigma) and 1 mg/mL Dispase (Stemcell Technologies) for 45 minutes at 37°C (200 rpm). Thereafter, the solution was filtered through 100 μm mesh, and the supernatant pelleted (1600 rpm, 5 minutes at 4°C), enriched through a percoll gradient (40 to 80%), and kidney leukocytes were collected and analyzed by FACS. Blood obtained from the retro-orbital sinus was immediately placed in heparinized tubes and washed with PBS followed by RBC lysis buffer (10 mM KHCO₃, 16 mM NH₄Cl, pH 7.3) prior to FACS analysis. Fluorophore-conjugated antibodies used for flow cytometry include B220 (RA3-6B2, BioLegend), CD4 (GK1.5, eBioscience), CD8 (53-6.7, eBioscience), CD11b (M1/70, BioLegend), CD45 (30-F11, eBioscience), Foxp3 (FJK-16S, eBioscience), IL-17A (eBio17B7, eBioscience), Ly6C (HK1.4, eBioscience), Ly6G (1A8, BD Biosciences), Ly6G (RB6-8C5, eBioscience), RORγT (AFKJS-9, eBioscience). For neutrophil depletion, 500 μg of purified anti-Ly6G (1A8, BioXcell) or isotype (Rat IgG2a, BioXcell) antibody was administered by intraperitoneal injection 10 days after C. albicans infection with sustained dosing every three days thereafter.

Jiang T.T., Chaturvedi V., Ertelt J.M., Xin L., Clark D.R., Kinder J.M, & Way S.S. (2014). Commensal enteric bacteria lipopolysaccharide impairs host defense against disseminated Candida albicans fungal infection. Mucosal immunology, 8(4), 886-895.

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Histological Analysis of Hepatic Inflammation

Cited 2 times

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Liver specimens (4μm), stained with hematoxylin and eosin (H&E), were analyzed blindly [21 (link), 22 (link)]. Primary mAb against mouse neutrophils (Ly-6G) (1A8; BD Biosciences, San Jose, CA) and macrophages (CD68) (FA-11; AbD Serotec, Raleigh, NC) were used. Labeled cells were counted in 10 high-power fields (HPF). Superoxide levels in hepatic tissue were detected with ROS-sensing dye dihydroethidium (DHE; AnaSpec, Fremont, CA).

Zhang C., Zhang Y., Liu Y., Liu Y., Kageyama S., Shen X.D., Gao F., Zheng S., Busuttil R.W., Shaw G.D., Ji H, & Kupiec-Weglinski J.W. (2017). A SOLUBLE FORM OF PSGL-1 REQUIRES NRF2 SIGNALING TO PROTECT LIVER TRANSPLANT ENDOTHELIAL CELLS AGAINST ISCHEMIA-REPERFUSION INJURY. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 17(6), 1462-1475.

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