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Ly6g clone 1a8

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Ly6G (clone 1A8) is a monoclonal antibody that binds specifically to the Ly6G antigen. Ly6G is a cell surface marker expressed on neutrophils. The antibody can be used for the identification and quantification of neutrophils in various applications.

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Lab products found in correlation

Cd11b clone m1 70, by BD (5 mentions) Cd45 clone 30 f11, by BD (4 mentions) Ly6c clone al 21, by BD (3 mentions) Cd11b clone m1 70, by BioLegend (3 mentions) Cd11b clone m1 70, by Thermo Fisher Scientific (3 mentions) Facscanto 2, by BD (3 mentions) Cd62l clone mel 14, by BD (2 mentions) Cd4 clone gk1, by BioLegend (2 mentions) Cd45.2 clone 104, by BD (2 mentions) Cd11c clone n418, by BioLegend (2 mentions) Cytofix cytoperm kit, by BD (2 mentions) Cd11b, by BD (2 mentions) F4 80 clone bm8, by Thermo Fisher Scientific (2 mentions) Live dead near infrared viability dye, by Thermo Fisher Scientific (1 mentions) Cd69 clone h1.2f3, by BD (1 mentions) Cd3 clone 17a2, by BioLegend (1 mentions) Fcs express software, by De Novo Software (1 mentions) Cd11c clone n418, by Thermo Fisher Scientific (1 mentions) Fcγ block, by Thermo Fisher Scientific (1 mentions) Cd45.1 clone a20, by BD (1 mentions)

Close spelling variants of this product

Ly6G-PE (clone 1A8, CD11b-PE and Ly6G-FITC clone 1A8 FITC-conjugated anti-Ly6G (clone 1A8, Anti-Ly6G-PE (clone 1A8) Rat monoclonal antibody to Ly6G ([Gr-1] clone 1A8; PE-conjugated anti-mouse Ly6G (clone 1A8) Purified Rat anti-mouse Ly6G (clone 1A8) Rat anti-Ly6G (clone 1A8, Ly6G-FITC (clone 1A8, Ly6G PerCP-Cy5.5 clone 1A8

21 protocols using ly6g clone 1a8

1

Immune Cell Profiling in Murine Super-Infection

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Whole blood was collected from mice on days −1, 3, 7 and 9 post‐super‐infection. Blood was collected in 0.5 M EDTA, then lysed using ACK Lysis buffer, washed and resuspended in flow cytometry buffer (2% FCS + 0.5 mM EDTA in PBS). Single‐cell suspensions were blocked with anti‐CD16/32 antibody and stained with fluorochrome‐conjugated antibodies against mouse CD3 (clone 17A2; Biolegend, San Diego, CA, USA), CD4 (clone GK1.5, Biolegend), CD8 (clone 53‐6.2, BD Biosciences, Franklin Lakes, NJ, USA), CD45 (clone 30‐F11, BD Biosciences), CD11b (clone M1/70, BD Biosciences), IA/IE (M5/114; eBioscience, San Diego, CA, USA), Ly6G (clone 1A8, BD Biosciences), CD62L (clone MEL‐14, BD Biosciences) and CD69 (clone H1.2F3, BD Biosciences), and dead cells were excluded using LIVE/DEAD Near‐Infrared viability dye (#L110909, Thermo Fisher Scientific). Sphero™ Blank Calibration Particles (BD Biosciences) were added to the samples and used as reference counting beads. Cells were acquired on a BD LSR II Fortessa Cell Analyser, and data were analysed using FlowJo software (v10.6; Treestar, Inc.).
Zaman M., Huber V.C., Heiden D.L., DeHaan K.N., Chandra S., Erickson D., Ozberk V., Pandey M., Bailly B., Martin G., Langshaw E.L., Zaid A., von Itzstein M, & Good M.F. (2021). Combinatorial liposomal peptide vaccine induces IgA and confers protection against influenza virus and bacterial super‐infection. Clinical & Translational Immunology, 10(9), e1337.
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2

Neutrophil Identification and Apoptosis

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Isolated lung cells were incubated with FITC-conjugated antibody to the neutrophil-specific marker Ly6G (clone 1A8, BD Pharmingen, San Diego, CA, USA) or the appropriate isotype control at 4°C in the dark. Cells were washed with staining buffer to remove unbound antibody. Labeling with Alexa Fluor 647-conjugated Annexin V and 7-AAD were performed according to the manufacturer’s instructions (BioLegend, San Diego, CA). Stained samples were analyzed using a CyAn ADP flow cytometer (Dako/Beckman-Coulter, Brea, CA, USA). Flow cytometry data were analyzed using FCS Express software (De Novo Software, Los Angeles, CA, USA).
Gomez J.C., Dang H., Martin J.R, & Doerschuk C.M. (2016). Nrf2 modulates host defense during S. pneumoniae pneumonia in mice. Journal of immunology (Baltimore, Md. : 1950), 197(7), 2864-2879.
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3

Phagocytosis of Staphylococcus aureus by BMDCs

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PECs or blood leukocytes were blocked in Fcγ block (1 μg/ml; eBioscience) and then surface stained with fluorochrome-conjugated antibodies against Ly6G (clone 1A8; BD Bioscience), F4/80 (clone BM8; eBioscience), CD11c (clone N418; eBioscience), and CD11b (clone M1/70; eBioscience). Flow cytometric data were acquired with a BD FACSCanto II instrument (BD Biosciences) and analyzed using FlowJo software (Tree Star).
To assess the rate of S. aureus phagocytosis by BMDCs, cells that had been infected with CTV-labeled S. aureus for 30 min or 2 h were incubated with gentamicin (200 μg/ml) for 1 h, washed, and fixed in 2% PFA. The cells were then analyzed on BD FACSCanto II by gating on forward scatter and side scatter, and the percentage of CTV+ cells was assessed.
O'Keeffe K.M., Wilk M.M., Leech J.M., Murphy A.G., Laabei M., Monk I.R., Massey R.C., Lindsay J.A., Foster T.J., Geoghegan J.A, & McLoughlin R.M. (2015). Manipulation of Autophagy in Phagocytes Facilitates Staphylococcus aureus Bloodstream Infection. Infection and Immunity, 83(9), 3445-3457.
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4

Isolation and Characterization of Lung Immune Cells

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BALF was recovered from mice at the specified timepoint in 1 ml sterile PBS containing 5 mM EDTA as previously described [90 (link)]. Cells were surface stained 30 min in cold FACS buffer (PBS with 2.5% FBS, 5 mM EDTA) with Siglec-F (clone E50-2440, Biolegend), CD11c (clone N418, Biolegend), Ly6G (clone 1A8, BD Biosciences), Ly6C (clone AL-21, BD Biosciences, Allschwil, Switzerland), CD11b (clone M1/70, Biolegend), CD45.1 (clone A20, BD Biosciences), CD45.2 (clone 104, BD Biosciences).
For intracellular staining of TNF (clone MP6-XT22, Biolegend), mice were injected i.p. with 50 μl of 5 mg/ml Brefeldin A in EtOH (diluted with 100 μl PBS) 3 hours prior to taking BALF. Lavage was performed with 1 ml PBS 5mM EDTA containing 5 μg/ml Brefeldin A, and was immediately placed on ice. After surface stain, cells were washed with FACS buffer and fixed, permeabilized and stained using the BD Biosciences Cytofix/Cytoperm Kit according to the manufacturer's instructions. Data were acquired on an LSRII (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, OR). An Aria III instrument (BD Biosciences) was used for cell sorting.
Ziltener P., Reinheckel T, & Oxenius A. (2016). Neutrophil and Alveolar Macrophage-Mediated Innate Immune Control of Legionella pneumophila Lung Infection via TNF and ROS. PLoS Pathogens, 12(4), e1005591.
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5

Characterization of Brain Immune Cells

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BILs were incubated with 0.5 μg Fc block (anti-FcyRIII/II mAb) prepared from the supernatant of 2.4G2 hybridoma cells for 30 min on ice, followed by staining with CD45 (clone 30-F11, BD Biosciences), CD11b (clone M1/70, BD Biosciences), Ly6G/C (clone RB6-8C5, BD Biosciences), or Ly6G (clone 1A8, BD Biosciences). All antibodies were added to blocked wells at 1:200, incubated for 30 min, and washed three times prior to flow cytometric analysis. Brain-infiltrating cells were gated on CD45 expression. All CD45mid/hi cells were further assessed for expression of CD11b, Ly-6C/G (Gr1), and Ly-6G (1A8). We defined inflammatory monocytes as the CD45hiCD11b+Gr1++1A8− population, neutrophils as CD45hiCD11b++Gr1+1A8+ cells, and microglia as CD45midCD11bmidGr1− cells. For phenotyping experiments involving reporter LysM:eGFP mice, we defined inflammatory monocytes as GFPmid cells, neutrophils as GFPhi cells, and microglia as GFPneg cells within a specific forward- and side-scatter gate. Flow cytometric analysis was performed on an Accuri C6 flow cytometer with sampler arm (BD Biosciences, Mountain View, CA). Files were analyzed offline using FlowJo 10.08 (Windows version; FlowJo LLC, Ashland, OR).
Howe C.L., LaFrance-Corey R.G., Goddery E.N., Johnson R.K, & Mirchia K. (2017). Neuronal CCL2 expression drives inflammatory monocyte infiltration into the brain during acute virus infection. Journal of Neuroinflammation, 14, 238.
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6

Lamina Propria Cell Immunophenotyping

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Isolated lamina propria cells were resuspended in PBS containing 1% FBS and stained with fluorescence-conjugated mAbs to CD4 (clone GK 1.5), CD45 (clone 30-F11), and Ly6G (clone 1A8) and isotype controls, purchased from BD Biosciences. F4/80 (clone Cl: A3–1) was obtained from Serotech. Abs were diluted at 1:100 when used. Stained cells were analyzed on an LSRFortessa X-20 cytometer and data were analyzed with FlowJo software.
Liu X., Lu J., Liu Z., Zhao J., Sun H., Wu N., Liu H., Liu W., Hu Z., Meng G., Shen L., Miller A.W., Su B., Li X, & Kang Z. (2018). Intestinal Epithelial Cell–Derived LKB1 Suppresses Colitogenic Microbiota. Journal of immunology (Baltimore, Md. : 1950), 200(5), 1889-1900.
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7

Multiparametric Flow Cytometry of Liver and Spleen

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For flow cytometric analysis, liver and spleen single-cell suspensions were stained using the following antibodies: Cd11c, clone N418 (Biolegend, San Diego, CA, 117339); Ly6c, clone HK1.4 (Biolegend, 128035); MHCII, clone M5/114.15.2 (eBioscience, Waltham, MA, 48-5321-82); Cd45, clone 30-F11 (eBioscience, 56-0451-82); Cd11b, clone M1/70 (eBioscience, 47-0112-82); Cd64, clone X54-5/7.1 (BD Biosciences, Franklin Lakes, NJ, 741024); Ly6g, clone 1A8 (BD Biosciences, 560601); Fc block Cd16/Cd32, clone 2.4G2 (BD Biosciences, 553142); Ly6g, RB6-8c5 (Tonbo, San Diego, CA, 60-5931); Ghost (Tonbo, 13-0870-T100); Flow cytometric data was acquired on the BD LSRII Fortessa X20 and analyzed using FlowJo (Franklin Lakes, NJ).
Alkhani A., Levy C.S., Tsui M., Rosenberg K.A., Polovina K., Mattis A.N., Mack M., Van Dyken S., Wang B.M., Maher J.J, & Nijagal A. (2020). Ly6cLo non-classical monocytes promote resolution of rhesus rotavirus-mediated perinatal hepatic inflammation. Scientific Reports, 10, 7165.
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8

Comprehensive Immune Cell Phenotyping

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Cell suspensions were stained in ice cold PBS with or without 2 % FBS on ice. The following antibodies were used for staining mouse cells: CD45 (clone 30-F11, Biolegend), Ly6G (clone 1A8, BD Biosciences), CD11c (clone HL3, BD Biosciences), CD11b (clone M1/70, Biolegend), Siglec F (clone E50–2440, BD Biosciences), CD3e (clone 145–2C11, eBioscience), CD4 (clone GK1.5, Biolegend), IFN © (clone XMG1.2, Biolegend), IL-17 (clone eBio17B7, eBioscience), IL-5 (clone TRFK5, Biolegend), IL-13 (clone eBio13A, eBioscience), CD25 (clone PC61.5, eBioscience), CD44, CD62L (clone MEL-14, BD Biosciences), MHC class II I-A/I-E (clone M5/114.15.2, eBioscience), DC-SIGN/CD209 (R&D systems), CD32 (clone D34–485, eBioscience), TLR4/CD284 (clone SA15–21, Biolegend), Annexin V (Biolegend). Neutrophils were defined as CD45+CD11b+Ly6G+DNA+ and cytoplasts were defined at CD45+CD11b+Ly6G+DNA−. Naïve T lymphocytes were defined as CD4+CD25−CD44lowCD62Lhi. Lung Dendritic cells were defined as CD45+CD11c+ MHCII+autofluorescencelow. The following antibodies were used to stain human cells: anti-CD45 PE-Cy7 (HI30), anti-CD66b (G10F5), anti-CD16 APC-Cy7 (3G8) all from Biolegend. Vybrant DyeCycle Ruby (Life Technologies) was used to stain intracellular DNA. Data were acquired on BD Canto II or BD LSR Fortessa and analyzed using FlowJo v10. For cell sorting, FACS Aria was used.
Krishnamoorthy N., Douda D.N., Brüggemann T.R., Ricklefs I., Duvall M.G., Abdulnour R.E., Martinod K., Tavares L., Wang X., Cernadas M., Israel E., Mauger D.T., Bleecker E.R., Castro M., Erzurum S.C., Gaston B.M., Jarjour N.N., Wenzel S., Dunican E., Fahy J.V., Irimia D., Wagner D.D, & Levy B.D. (2018). Neutrophil cytoplasts induce Th17 differentiation and skew inflammation toward neutrophilia in severe asthma. Science immunology, 3(26), eaao4747.
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9

Quantification of Lung Inflammation in Scald Injury

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As described above, BAL fluid was isolated from sham, scald-injured, and MV treated scald-injured mice and centrifuged at 450 × g for 10 minutes at room temperature. Cell free BAL fluid was used to determine IL-6 (BD Biosciences) levels by cytometric bead array, performed according to the manufacturer’s protocol. Cells were quantified using a Coulter AcT 10 cell counter (Beckman Coulter, Brea, CA) and then suspended in FACS buffer (PBS with 1% bovine albumin and 0.1% azide) and Fc Block (BD Pharmingen, San Jose, CA). Neutrophils were characterized by staining with Ly-6G (clone 1A8, BD Biosciences) and CD11b (clone M1/70, BD biosciences) antibodies. Cells were analyzed on Attune Acoustic Focusing Cytometer using Attune Cytometric Software v2 (Thermo Fisher Scientific).
Rice T.C., Pugh A.M., Xia B.T., Seitz A.P., Whitacre B.E., Gulbins E, & Caldwell C.C. (2017). Bronchoalveolar Lavage Microvesicles Protect Burn-Injured Mice from Pulmonary Infection. Journal of the American College of Surgeons, 225(4), 538-547.
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10

Ear Pinna Immune Cell Isolation

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Ear pinna dermal sheets were harvested, treated with ethanol, separated using forceps and digested in PBS containing Liberase TL (Roche) at 37°C for 1 h. Digested tissues were homogenized in a tissue homogenizer (Medimachine; BD biosciences) and filtered in a 50 µm strainer. The resulting single cell suspensions were stained for Ly6C (clone AL-21; FITC; BD), Ly6G (clone 1A8; PE; BD), CD11b (clone M1/70; PE-Cy7; BD), and with the Fixable Yellow Dead Cell Stain Kit (Invitrogen), after being incubated with anti-Fc (CD16/32) antibodies to block unspecific binding for 30 min. For intracellular detection of IL-1β, ear cells were blocked and stained with surface markers as mentioned above. Thereafter, cells were fixed and permeabilized with Cytofix/Cytoperm (BD), followed by incubation with anti-mouse IL-1β proform antibody (clone NJTEN3, APC; eBioscience). Data were analyzed on a MACSQuant flow cytometer (Miltenyi Biotec). Cells were acquired based on forward and side scatter and data analyzed with FlowJo Software 4.3.
Dey R., Joshi A.B., Oliveira F., Pereira L., Guimarães-Costa A.B., Serafim T.D., de Castro W., Coutinho-Abreu I.V., Bhattacharya P., Townsend S., Aslan H., Perkins A., Karmakar S., Ismail N., Karetnick M., Meneses C., Duncan R., Nakhasi H.L., Valenzuela J.G, & Kamhawi S. (2017). Gut Microbes Egested During Bites of Infected Sand flies Augment Severity of Leishmaniasis Via Inflammasome-Derived IL-1β. Cell host & microbe, 23(1), 134-143.e6.
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