Ly6g 1a8

Manufactured by BioLegend

Sourced in United States

Ly6G (1A8) is a monoclonal antibody that recognizes the Ly6G antigen. Ly6G is a glycosylphosphatidylinositol (GPI)-linked cell surface protein primarily expressed on neutrophils. The antibody can be used for the identification and characterization of neutrophils in flow cytometry and other immunoassays.

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Spelling variants of this product

Ly6G (clone 1A8, (49 citations) Anti-Ly6G-APC (clone 1A8, (11 citations) Ly6G-PE (1A8, (7 citations) Ly6G-APC (1A8; (4 citations) APC anti-mouse Ly6G (Clone 1A8) (4 citations) Ly6G‐PerCPCy5.5 (clone 1A8 (4 citations) Anti-mouse Ly6G (clone 1A8, (3 citations) Ly6G-PE (clone 1A8) (3 citations) PE)-conjugated anti-mouse Ly6G antibody (clone 1A8), (2 citations) Anti-Ly6G-APC/Cy7 (clone 1A8) (2 citations)

66 protocols using ly6g 1a8

Multiparametric Immune Cell Profiling

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Peripheral blood was collected retro-orbitally from isoflurane-anesthetized mice. The blood was treated with ACK lysis buffer for 5 min to remove RBCs, leaving the peripheral blood leukocytes (PBLs). To determine expression of cell surface molecules, we incubated PBLs with mAb at 4°C for 20–30 min, and cells were subsequently fixed for 10 min using Cytofix Solution (BD Biosciences). The following mAb clones were used to stain processed samples: CD11a (M17/4; eBioscience), TLR4 (SA15-21; BioLegend), CD3ε (145-2C11; BioLegend), NK1.1 (PK136; eBioscience), NKp46 (29A1.4; BioLegend), F4/80 (BM8; BioLegend), TLR2 (CB225; BD Bioscience), CD19 (6D5; BioLegend), CD11c (HL3; BD Biosciences), CD4 (GK1.5; BioLegend), Ly6G (1A8; BioLegend), CD127 (A7R34; BioLegend), Ly6C (HK1.4; BioLegend), and CD8α (53-6.7; BioLegend). Flow cytometry data were acquired on a Cytek Aurora (Cytek, Bethesda, MD) and analyzed with FlowJo software (Tree Star, Ashland, OR).

Berton R.R., Jensen I.J., Harty J.T., Griffith T.S, & Badovinac V.P. (2022). Inflammation Controls Susceptibility of Immune-Experienced Mice to Sepsis. ImmunoHorizons, 6(7), 528-542.

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Immunofluorescence Analysis of FRT Tissue

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FRT tissues were embedded in Tissue-Tek OCT (Sakura, Netherlands). Sections were fixed with acetone, and blocked (50 μg/ml mouse IgG and 10% BSA) and stained with: Ly6g 1A8 (Biolegend), F4/80 BM8 (Biolegend), MHCII 2G9 (BD Biosciences), CD31 2H8 (Invitrogen), α-SMA 1A4 (eBioscience), CD62-E (BD Bioscience), CD62P REA344 (Miltenyi Biotec), ICAM-1 M-19 (Santa Cruz Biotechnology), CD106 429 (Miltenyi Biotec). For protein expression quantification, tissues were imaged using the glycerol ACS APO 20x NA 0.60 objective of a confocal fluorescence microscope (SPE, Leica Microsystems), maintaining the acquisition settings all over the process for each sample and among samples, as previously described (19 (link)). Mean fluorescence intensities (MFI) were assessed at multiple regions of interest (ROIs) by field, using the glycerol ACS APO 63x objective and randomly depicted at specific areas of the venular beds in FRT. All quantifications were performed using the FIJI (Fiji Is Just ImageJ) software (NIH).

Latorre M.C., Gómez‐Oro C., Olivera‐Valle I., Blazquez‐Lopez E., Gallego‐Valle J., Ibañez‐Escribano A., Casesnoves P., González‐Cucharero C., Muñoz‐Fernandez M.A., Sanz L., Vaquero J., Martín‐Rabadań P., Perez‐Milan F, & Relloso M. (2022). Vaginal neutrophil infiltration is contingent on ovarian cycle phase and independent of pathogen infection. Frontiers in Immunology, 13, 1031941.

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Multiparameter Flow Cytometry of Tumor Cells

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Tumor tissues were digested both mechanically by chopping with razor blades and chemically with 1mg/mL type IA collagenase (Sigma-Aldrich) for 30 minutes at 37°C. Following digestion, cell suspensions were washed, filtered and stained as previously described (O’Sullivan et al., 2012 (link)). The following antibodies were used: Ly6C (ER-MP20, Serotec), MHCII (M5/114 15.2, eBioscience), Ly6G (1A8, Biolegend), CD8 (53-6.7, eBioscience), CD44 (IM7, Biolegend), CD3 (17A.2, Biolegend), CD4 (GK1.5, Biolegend), CD69 (H1.2F3, Biolegend), Granzyme B (NGZB, eBioscience), IFNγ (XMG 1.2, Biolegend), TCRβ (H57-597, Biolegend), B220 (RA3-6B2, eBioscience), NK1.1 (PK136, Biolegend), CD11b (M1/70, eBioscience), CD45 (30-F11, Biolegend). Stained cell suspensions were analyzed on a BD FACS CANTO II (BD Biosciences).

Saddawi-Konefka R., Seelige R., Gross E.T., Levy E., Searles S.C., Washington A J.r., Santosa E.K., Liu B., O’Sullivan T.E., Harismendy O, & Bui J.D. (2016). Nrf2 induces IL-17D to mediate tumor and virus surveillance. Cell reports, 16(9), 2348-2358.

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Characterization of BAL Neutrophils

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Mouse BAL cells were treated with α-CD16/32 Fc block (eBioscience) and mouse serum (Thermo Fisher Scientific) prior to staining with antibodies. Relevant full minus one (FMO) samples for each group were used as controls. Antibodies used were as follows: Ly6G (1A8, BioLegend), CD11a (M17/4, BioLegend), CD11b (M1/70, BioLegend), CXCR2 (SA044G4, BioLegend), and CXCR1 (FAB8628A-025, RnD). Live cells were gated following staining with DAPI (Invitrogen) prior to acquisition. BAL neutrophils were gated according to Ly6G⁺ and forward scatter (FSC)/side scatter (SSC) properties. Cells were acquired on an LSRFortessa (BD). Compensation was performed using BD FACSDiva software and data analyzed with FlowJo version 10.

Sadiku P., Willson J.A., Dickinson R.S., Murphy F., Harris A.J., Lewis A., Sammut D., Mirchandani A.S., Ryan E., Watts E.R., Thompson A.A., Marriott H.M., Dockrell D.H., Taylor C.T., Schneider M., Maxwell P.H., Chilvers E.R., Mazzone M., Moral V., Pugh C.W., Ratcliffe P.J., Schofield C.J., Ghesquiere B., Carmeliet P., Whyte M.K, & Walmsley S.R. (2017). Prolyl hydroxylase 2 inactivation enhances glycogen storage and promotes excessive neutrophilic responses. The Journal of Clinical Investigation, 127(9), 3407-3420.

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Cryosectioning and Immunofluorescence Analysis

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Immunofluorescence microscopy in cryosections: After overnight fixation in PFA4%, hearts were embedded in Tissue-Tec (Sakura) and immediately frozen at −80°C. Frozen blocks were cut into 30 µm sections and mounted on poly-L-lysine-coated glass slides (Menzel, Braunschweig, Germany). We used the following antibodies from eBiosciences and Bio Legend: CD31 (MEC 13.3), F4/80 (BM-8) and Ly6G (1A8). Furthermore we utilized AF488 labeled rabbit anti-GFP antibody purchased by Life technologies (Carlsbad, CA) and costained the slices with a donkey anti-rabbit antibody in AF647 (Life technologies) to minimize backgound fluorescence. Slices were analysed by immune fluorescence confocal microscopes (LSM 510 Meta and LSM 780, Carl Zeiss Microscopy GmbH, Jena, Germany). Data was analyzed with Imaris-software (Bitplane AG Zurich, Switzerland).

Weisheit C., Zhang Y., Faron A., Köpke O., Weisheit G., Steinsträsser A., Frede S., Meyer R., Boehm O., Hoeft A., Kurts C, & Baumgarten G. (2014). Ly6Clow and Not Ly6Chigh Macrophages Accumulate First in the Heart in a Model of Murine Pressure-Overload. PLoS ONE, 9(11), e112710.

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Characterization of Lung Immune Cells

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Cells were harvested from whole lungs and/or broncheoalveolar lavage and flow cytometry was performed as we have previously reported using standard techniques for labeling of cells (19 (link), 22 (link), 26 (link)). Stained cells were analyzed with a FACSCalibur flow cytometer (BD) and data analyzed with FlowJo software (Tree Star, Inc). For FACS, a FACSAria cell sorter (BD Biosciences, USA) was used to identify and collect neutrophils. The following antibodies were used for flow cytometry experiments: Ly6G (1A8); CD11c (N418); Gr-1 (RB6-8C5); all from BioLegend.
Neutrophil and PMN-DC hybrid were identified as follows by flow cytometry: PMN-DC hybrid (CD11c⁺ Ly6G⁺), PMN (CD11c⁻ Ly6G⁺) and same gating also for sorting. Neutrophils for FACS were gated as (singlets, CD45.2⁺, Ly6G⁺).

Hussain S.R., Rohlfing M., Resiliac J., Santoro J., Peeples M.E., Garcin D, & Grayson M.H. (2021). Atopic neutrophils prevent post-viral airway disease. Journal of immunology (Baltimore, Md. : 1950), 207(10), 2589-2597.

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Multicolor Flow Cytometry Immunophenotyping

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For flow cytometry or cell sorting, cells were transferred to 15-mL tubes and washed in cold buffer (2% FCS, 2 mM EDTA in PBS). Before antibody labeling, cells were incubated with Fc-Block (1:200, anti-CD16/32 antibody; 2.4G2, BD Pharmingen) for 10 min at 4°C and washed. Cells were stained in buffer with (1:100) fluorescently labeled antibodies against CD11b (clone M1/70, BD Pharmingen), CD11c (clone N418, Biolegend), CD115 (clone AFS98, eBioscience), CD19 (clone 1D3, BD Pharmingen), and Ly-6G (1A8, Biolegend) for 30 min at 4°C in the dark. 7-AAD (BD Pharmingen) was added before measurement to exclude dead cells. Analysis was performed on LSRFortessa and sorting on FACSAria II or III (BD Pharmingen). Unstained, empty vector-transduced cells or fluorescence-minus-one staining setups served as controls.

Cirovic B., Schönheit J., Kowenz-Leutz E., Ivanovska J., Klement C., Pronina N., Bégay V, & Leutz A. (2017). C/EBP-Induced Transdifferentiation Reveals Granulocyte-Macrophage Precursor-like Plasticity of B Cells. Stem Cell Reports, 8(2), 346-359.

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Characterizing Immune Cells in Arthritic Mice

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CAIA mice were intravenously injected with control IgG, unmodified α-TNF, or CBP–α-TNF at a dose of 200 μg per mouse. On day 5, hind paws were harvested and digested in Dulbecco’s modified eagle medium supplemented with 2% FBS, collagenase D (2 mg/ml) and deoxyribonuclease I (40 μg/ml; Roche) for 30 min at 37°C. Single-cell suspensions were obtained by gently disrupting through a 70-μm cell strainer. Antibodies against the following molecules were used: anti-mouse CD45 (30-F11, BD Biosciences), F4/80 (T45-2342, BD Biosciences), Ly6G (1A8, BioLegend), Ly6C (HK1.4, BioLegend), and CD11b (M1/70, BioLegend). Fixable live/dead cell discrimination was performed using Fixable Viability Dye eFluor 455 (eBioscience) according to the manufacturer’s instructions. Following a washing step, cells were stained with specific antibodies for 20 min on ice. Flow cytometric analyses were done using a Fortessa (BD Biosciences) flow cytometer and analyzed using FlowJo software (Tree Star).

Katsumata K., Ishihara J., Mansurov A., Ishihara A., Raczy M.M., Yuba E, & Hubbell J.A. (2019). Targeting inflammatory sites through collagen affinity enhances the therapeutic efficacy of anti-inflammatory antibodies. Science Advances, 5(11), eaay1971.

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Immune Response Analysis in Malaria Infection

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Blood was harvested 6 d post infection with P. berghei. Red blood cells were lysed with ACK and leukocytes were incubated with LPS (100 ng/μL) or vehicle plus GolgiPlug (BD) and monensin (eBioscience) for 4 h. Samples were blocked with anti-Fc receptor antibody (α-CD16/32, clone 2.4G2; UCSF Monoclonal Antibody Core) and labeled with antibodies to CD11b (M1/70) and F4/80 (BM8) (UCSF), Ly6c (HK1.4; eBioscience) and Ly6g (1A8; BioLegend). Following fixation and permeablization with Cytofix/Cytoperm (BD), intracellular labeling was performed with antibodies to IL-1β (NJTEN3), TNF (TN3-19), or rat IgG1 isotype (eBRG1) (all eBioscience). Samples were analyzed on an LSR II (BD). Classical monocytes were defined as CD11b⁺ Ly6g⁻ F4/80^int Ly6c^hi, as previously described (13 (link)).

Fontana M.F., Baccarella A., Kellar D., Oniskey T.K., Terinate P., Rosenberg S.D., Huang E.J., Herbert D.R, & Kim C.C. (2015). Myeloid expression of the AP-1 transcription factor JUNB modulates outcomes of type 1 and type 2 parasitic infections. Parasite immunology, 37(9), 470-478.

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Tissue Sectioning and Immunofluorescence Staining

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Tissue was embedded in optimal cutting temperature medium (TissueTec) and directly snap frozen in isopentane cooled by dry ice. The tissue was then cut into 8- or 14-µm sections by a cryostat and mounted on superfrost glass slides, which were dried for at least 1 h before storage at −70°C. Tissue sections were fixed and permeabilized with ice-cold acetone. The following antibodies were used: B220 (RA3-6B2; BioLegend), CD3 (pc; Abcam), CD68 (FA-11; AbD Serotec), CD103 (2E7; BioLegend), CD138 (281-2; BioLegend), F4/80 (BM8; AbD Serotec), IgG (STAR120F; AbD Serotec), Ly-6G (1A8; BioLegend), MARCO (ED31; AbD Serotec), MOMA1 (Abcam), and PNA (Vector). Tissue was mounted with Prolong Gold mountant (Invitrogen), and images were captured using a confocal scanning microscope (DMI6000; Leica Biosystems) connected to a True Confocal scanner (SP5; Leica Biosystems).

Parsa R., Lund H., Georgoudaki A.M., Zhang X.M., Ortlieb Guerreiro-Cacais A., Grommisch D., Warnecke A., Croxford A.L., Jagodic M., Becher B., Karlsson M.C, & Harris R.A. (2016). BAFF-secreting neutrophils drive plasma cell responses during emergency granulopoiesis. The Journal of Experimental Medicine, 213(8), 1537-1553.

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