Anti ly6g percp cy5

Manufactured by BioLegend

Sourced in United States

Anti-Ly6G-PerCP-Cy5.5 is a fluorescent-conjugated monoclonal antibody that binds to the Ly6G antigen. Ly6G is a cell surface marker expressed on mature granulocytes, including neutrophils. This product can be used for the identification and quantification of granulocytes in flow cytometry applications.

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10 protocols using anti ly6g percp cy5

Comprehensive MAIT and NKT Cell Phenotyping

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From each group of animals, 1–2 million cells aliquots were prepared and to exclude dead cells from analysis, cells were first stained with the fixable viability dye eFluor 780 (eBioscience) for 15 min at room temperature (RT). Cells were incubated with anti-mouse CD16/CD32 Fc Block antibody (BD Biosciences, San Jose, CA), for 20 min at 4°C. Cells were then stained for 30 min at RT with appropriately diluted PE-conjugated 5-OP-RU-loaded species-specific MR1-tetramers or α-GalCer (PBS-44)–loaded CD1d tetramer conjugated to APC, anti-CD3-FITC (BioLegend), anti-CD161-BV510 (BioLegend), anti-CD49b-BV711 (BD), anti-TCRγδ-PE-Cy7 (BioLegend), anti-TCRβ-BV421 (BioLegend), anti-CD45R-PE-Cy5, and anti-CD44-BV650 (BioLegend). To evaluate different antigen-presenting cells, after Fc Block incubation, cells were also surface stained with anti-CD45 AF700 (BioLegend), anti-CD11b-FITC (BioLegend), anti-CD11c-PE Cy-7 (BD), anti-Siglec-F- BV711 (BD), anti-CD64-BV605 (BioLegend), anti-CD24-PE (BioLegend), anti-Ly-6G-PerCP-Cy5.5 (BioLegend), anti-CD103-BV510 (BioLegend), anti-CD86-APC (BioLegend), anti-Ly6C (PE dazzle 594), and anti-MHC II BV421 (BioLegend) for 30 min at 4°C. Total 10⁶ gated events per sample were collected using Fortessa flow cytometer (Becton Dickinson, San Diego, CA), and results were analyzed using FlowJo 10.4.2 software.

Trivedi S., Labuz D., Anderson C.P., Araujo C.V., Blair A., Middleton E.A., Jensen O., Tran A., Mulvey M.A., Campbell R.A., Hale J.S., Rondina M.T, & Leung D.T. (2020). Mucosal-associated invariant T (MAIT) cells mediate protective host responses in sepsis. eLife, 9, e55615.

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Immune Cell Identification in Mouse BALF and Lung

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For the identification of immune cell populations, mouse BALF and lung cells were stained in the presence of counting beads (15.45 μm DragonGreen, Bangs Laboratories Inc., FS07F) with LIVE/DEAD stain (Invitrogen, L23105A) and an antibody mixture of anti-CD45-AF700 (BioLegend, 103127), anti-CD11b-AF594 (BioLegend, 101254), anti-CD11c-Bv605 (BioLegend, 117334), anti-SiglecF-AF647 (BD, 562680), anti-MHCII-APC Cy7 (BioLegend, 107628), anti-Ly6C-Bv421 (BioLegend, 128032), and anti-Ly6G-PerCP-Cy5.5 (BioLegend #127616), each at a concentration of 1:200 in PBS, for 1 h at 4°C. After washing, the cells were stored in 2% paraformaldehyde (PFA, Electron Microscopy Sciences, 15714-S) until analysis on the BD LSRII (BD Biosciences) using FACSDiva v9. Flow cytometry was analyzed with FlowJo v10. Mouse BAL and lung cells were identified as follows:
alveolar macrophages: CD45⁺CD11b^+/−SiglecF⁺CD11c⁺;
monocytes: CD45⁺CD11b⁺SiglecF⁻MHCII⁻CD11c⁻Ly6G⁻Ly6C^+/−;
neutrophils: CD45⁺CD11b⁺SiglecF⁻ MHCII⁻CD11c⁻Ly6G⁺Ly6C^+/−.

Wong Fok Lung T., Charytonowicz D., Beaumont K.G., Shah S.S., Sridhar S.H., Gorrie C.L., Mu A., Hofstaedter C.E., Varisco D., McConville T.H., Drikic M., Fowler B., Urso A., Shi W., Fucich D., Annavajhala M.K., Khan I.N., Oussenko I., Francoeur N., Smith M.L., Stockwell B.R., Lewis I.A., Hachani A., Baskota S.U., Uhlemann A.C., Ahn D., Ernst R.K., Howden B.P., Sebra R, & Prince A. (2022). Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection. Cell metabolism, 34(5), 761-774.e9.

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Isolation and Characterization of Murine Tumor Cells

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Single cell suspensions from murine primary pancreatic tumors and pulmonary metastasis were prepared by mechanical and enzymatic disruption and tumor cells, tumor associated macrophages and stromal cells were analyzed and sorted using flow cytometry (FACS ARIA II, BD Bioscience). Samples were digested as outlined above, the cells were then filtered through a 70 μm cell strainer and resuspended in PBS + 1% BSA, blocked for 10 min on ice with FC Block (BD Pharmingen, Clone 2.4G2) and stained with Sytox® blue viability marker (Life Technologies) and conjugated antibodies anti-CD45-PE/Cy7 (Biolegend, clone 30-F11) and anti-F4/80-APC (Biolegend, clone BM8).
Blood was collected from mice via tail vein bleed in EDTA-tubes. Red blood cell lysis was performed and resulting leukocytes were resuspended in PBS + 1% BSA and blocked for 10 min on ice with FC Block and stained with Sytox® blue viability marker and conjugated antibodies anti-CD45-APC/Cy7 (Biolegend, 103115), anti-CD11b-APC (Biolegend, 101212), anti-Ly6G-PerCP-Cy5.5 (Biolegend, 127616), anti-Ly6C-PE (Biolegend, 128008), anti-CD3-PE-Cy7 (Biolegend, 100320), anti-CD4-PE (Biolegend, 100408), and anti-CD8-PerCP-Cy5.5 (Biolegend, 100734). Cell analysis was performed using FACS Canto II.

Ireland L., Luckett T., Schmid M.C, & Mielgo A. (2020). Blockade of Stromal Gas6 Alters Cancer Cell Plasticity, Activates NK Cells, and Inhibits Pancreatic Cancer Metastasis. Frontiers in Immunology, 11, 297.

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Multicolor Flow Cytometry and Molecular Analyses of Immune Cells

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For flow cytometric analysis, anti-CD45.2-V500(562129, BD bioscience, 1:100), anti-CD45.2-APC(109814, BioLegend, 1:100), anti-CD4-PerCP/Cy5.5(100433, BioLegend, 1:100), anti-CD8a-PerCP/Cy5.5(100733, BioLegend, 1:100), anti-CD11b-PerCP/Cy5.5(45-0112-80, eBioscience, 1:100), anti-Ly6G-PerCP/Cy5.5(127615, BioLegend, 1:100), anti-B220-PerCP/Cy5.5(103235, BioLegend, 1:100), anti-CD11b-BV421(101251, BioLegend, 1:100), anti-CD64-APC(139306, BioLegend, 1:100), anti-F4/80-PE(123110, BioLegend, 1:100), anti-Ly6c-PE-Cy7(128018, BioLegend, 1:100) were used. For western blotting, anti-Cx40(AB1726, Merck Millipore, 1:500), anti-Cx43(C6219, Sigma, 1:2000), anti-Cx45(AB1745, Merck Millipore, 1:2000), anti- α -tubulin(T6199, Sigma, 1:1000), anti-rabbit IgG-HRP(7074, Cell Signaling Technology, 1:5000), anti-mouse IgG-HRP(7076, Cell Signaling Technology, 1:5000) were used. For immunohistochemistry, anti-F4/80(MCA497G, Serotec, 1:500), anti-Ly6G(127601, BioLegend, 1:500), anti-B220 (103201, BioLegend 1:500), anti-CD3(GTX42110, GeneTex, 1:500), anti-Cx43(C6219, Sigma, 1:2000), anti-N-cadherin(33-3900, ThermoFisher, 1:500) were used. The second antibodies used were Alexa-Fluor-488-conjugated anti-mouse-IgG(A-11001, ThermoFisher, 1:2000) and Alexa-Fluor-635-conjugated anti-rabbit-IgG(A-31576, ThermoFisher, 1:2000).

Sugita J., Fujiu K., Nakayama Y., Matsubara T., Matsuda J., Oshima T., Liu Y., Maru Y., Hasumi E., Kojima T., Seno H., Asano K., Ishijima A., Tomii N., Yamazaki M., Kudo F., Sakuma I., Nagai R., Manabe I, & Komuro I. (2021). Cardiac macrophages prevent sudden death during heart stress. Nature Communications, 12, 1910.

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Characterization of 4-1BBL and Calreticulin Expression in Osteosarcoma

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Viral 41BBL expression in osteosarcoma cell lines was determined by flow cytometry. Cells were stained first with a cell death detection antibody in the Zombie-NIR Fixable Viability Kit (423105, BioLegend) following the manufacturer's protocol and then with a PE-conjugated anti–4-1BBL antibody.
Calreticulin cell surface expression was determined by flow cytometry. Cells were stained first with a cell death detection antibody in the Zombie Green Fixable Viability Kit (423111, BioLegend) and then with a fluorophore-conjugated anti-calreticulin antibody (Abcam).
Fluorescence emission was analyzed using a FACSCanto II system with FACSDiva software (RRID:SCR_001456).
To identify immune cell populations, infiltrating immune cells were surface stained with the following antibody panel: anti–CD45-AF700 (BioLegend), anti–Ly6G-PerCP-Cy5.5 (BioLegend), anti–Ly6C-FITC(BioLegend), anti–F4/80-APC (BioLegend), anti–CD8-BV510 (BioLegend), anti–CD11b-BUV395 (BioLegend), and anti–CD4-BUV496 (BioLegend). PromoFLuor840 maleimide (PromoKine) was used as a viability marker. Samples were acquired with a CytoFlex flow cytometer (Beckman Coulter RRID:SCR_019627) and data analyses were performed using FlowJo v10 (RRID:SCR_008520).

Martinez-Velez N., Laspidea V., Zalacain M., Labiano S., García-Moure M., Puigdelloses M., Marrodan L., Gonzalez-Huarriz M., Herrador G., de la Nava D., Ausejo-Mauleon I., Fueyo J., Gomez-Manzano C., Patiño-García A, & Alonso M.M. (2022). Local Treatment of a Pediatric Osteosarcoma Model with a 4-1BBL Armed Oncolytic Adenovirus Results in an Antitumor Effect and Leads to Immune Memory. Molecular Cancer Therapeutics, 21(3), 471-480.

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Isolation and Identification of Myeloid Cells

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Tumors were minced and incubated for 30 min at 37°C in 2 mL digestion buffer (1 mg/mL collagenase (C2674, Sigma-Aldrich) and 100 μg/mL DNase I (D4527, Sigma-Aldrich) in RPMI 1640 medium). Cell suspensions were passed through a 100 μm cell strainer. After waching with RPMI 1640, cells were resuspended in 40% Percoll (17089101, Cytiva) and centrifuged. After centrifugation, Cells were washed with staining buffer.Spleens were minced and rinsed with 5 mM EDTA in RPMI 1640. Peripheral blood were obtained from mouse eyeballs and treated with anticoagulant reagent (G0280, Solarbio). Erythrocytes were lysed with 1 mL of ACK Lysing Buffer (C3702, Beyotime) per spleen for 2 min. Splenocytes and PBMCs were washed with RPMI 1640, centrifuged, followed by rinsing with staining buffer. After centrifugation, cells were resuspended in PBS at 1 × 10⁸ cells/mL and incubated with anti-mouse CD16/32 antibody (101302, Biolegend) and viability dye for 15 min at RT. Then, cells were stained with anti-CD11b-APC, anti-Ly-6C-FITC (128006, Biolegend) and anti-Ly-6G-PerCP/Cy5.5 (127616, Biolegend) antibody.

Wang Y., Sun Y., Deng S., Liu J., Yu J., Chi H., Han X., Zhang Y., Shi J., Wang Y., Quan Y., Li H, & Xu J. (2024). Discovery of galectin-8 as an LILRB4 ligand driving M-MDSCs defines a class of antibodies to fight solid tumors. Cell Reports Medicine, 5(1), 101374.

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Flow Cytometry Analysis of Osteosarcoma Cells

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Viral 41BBL expression in osteosarcoma cell lines was determined by flow cytometry. Cells were stained first with a cell death detection antibody in the Zombie-NIR™ Fixable Viability Kit (423105, Biolegend, San Diego, CA) following the manufacturer’s protocol and then with a PE-conjugated anti-4-1BBL antibody.
Calreticulin cell surface expression was determined by flow cytometry. Cells were stained first with a cell death detection antibody in the Zombie Green Fixable Viability Kit (423111, Biolegend, San Diego, CA) and then with a fluorophore-conjugated anti-calreticulin antibody (Abcam).
Fluorescence emission was analyzed using a FACSCanto™ II system with FACSDiva software (RRID:SCR_001456).
To identify immune cell populations, infiltrating immune cells were surface stained with the following antibody panel: anti-CD45-AF700 (Biolegend, San Diego, CA), anti-Ly6G-PerCP-Cy5.5 (Biolegend, San Diego, CA), anti-Ly6C-FITC (Biolegend, San Diego, CA), anti-F4/80-APC (Biolegend, San Diego, CA), anti-CD8-BV510 (Biolegend, San Diego, CA), anti-CD11b-BUV395 (Biolegend, San Diego, CA), and anti-CD4-BUV496 (Biolegend, San Diego, CA). PromoFLuor840 maleimide (PromoKine) was used as a viability marker. Samples were acquired with a CytoFlex flow cytometer (Beckman Coulter RRID:SCR_019627) and data analyses were performed using FlowJo v10 (RRID:SCR_008520).

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Multiparametric Flow Cytometry Analysis

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For flow cytometry analysis, isolated cells were pre-incubated for 10 minutes with purified anti-mouse CD16/32 (BioLegend) to block the Fc-mediated non-specific binding of antibodies. Then, cells were stained with the following antibodies: anti-CD64-PE/Dazzle 594, anti-CD115-PE/Dazzle 594, anti-CD3-PerCP/Cy5.5, anti-NK1.1-PerCP/Cy5.5, anti-Ly6G-PerCP/Cy5.5, anti-CD24-PE/Cy7, anti-CX3CR1-APC, anti-CCR5-APC, anti-Ly6C-APC/Cy7, anti-CD45-BV421, anti-I-A/I-E-BV605 (all from BioLegend), anti-CD19-PerCP/Cy5.5, anti-CD11b-AF700 (BD Biosciences, Franklin Lakes, NJ, USA), anti-CCR2-APC (R&D Systems, Minneapolis, MN, USA), and anti-F4/80-PE (Thermo Fisher Scientific) for 20 minutes on ice. After surface staining, dead cells were stained with Zombie aqua™ Fixable Viability Kit (BioLegend). For the intracellular staining of TLR7, cells were fixed and permeabilized with the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) according to the manufacturer’s instructions. Then, cells were stained with anti-TLR7-PE (BD Biosciences). Data were acquired on a FACS LSR Fortessa (BD Biosciences) and the percentage of each cell population and mean fluorescence intensity were analyzed using FlowJo software (TreeStar Inc, Ashland, OR, USA).

Nomura A., Mizuno M., Noto D., Aoyama A., Kuga T., Murayama G., Chiba A, & Miyake S. (2022). Different Spatial and Temporal Roles of Monocytes and Monocyte-Derived Cells in the Pathogenesis of an Imiquimod Induced Lupus Model. Frontiers in Immunology, 13, 764557.

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Murine Spinal Cord Leukocyte Profiling

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Spinal cord was collected from euthanized mice after systemic perfusion and Myelin Removal Beads (Miltenyi Biotec) were applied to obtained leukocyte-enriched fraction.
Following antibodies were used: anti-CD45-PE, anti-CD11b-AF488 and anti-Siglec-F-AF647 (BD Biosciences); anti-CCR3-PE-Vio770, anti-CD4-APC, anti-CD8-PerCP (Miltenyi Biotec); anti-Ly6G-PerCP/Cy5.5 (BioLegend); anti-F4/80-PE/Cy7 (eBioscience). FACSCanto II and FlowJo software were used for analysis.

Ruppova K., Lim J.H., Fodelianaki G., August A, & Neuwirth A. (2021). Eosinophils are dispensable for development of MOG_35-55-induced experimental autoimmune encephalomyelitis in mice. Immunology letters, 239.

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Multiparametric Flow Cytometry Analysis

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From each group of animals, 1-2 million cells aliquots were prepared and to exclude dead cells from analysis cells were first stained with the fixable viability dye eFluor TM 780 (eBioscience) for 15 min. at room temperature (RT). Cells were incubated with antimouse CD16/CD32 Fc Block antibody (BD Biosciences, USA), for 20 min at 4 °C. Cells were then stained for 30 min at RT with appropriately diluted PE conjugated 5-OP-RUloaded species-specific MR1-tetramers or α-GalCer (PBS-44)-loaded CD1d tetramer conjugated to APC, anti-CD3-FITC (Biolegend), anti-CD161-BV510 (Biolegend), anti-CD49b-BV711 (BD), anti-TCRγδ-PE-Cy7 (Biolegend), anti-TCRβ-BV421 (Biolegend), anti-CD45R-PE-Cy5 and anti-CD44-BV650 (Biolegend). To evaluate different antigen presenting cells, after Fc Block incubation, cells were also surface stained with anti-CD45 AF700 (Biolegend), anti-CD11b-FITC (Biolegend), anti-CD11c-PE Cy-7 (BD), anti-Siglec-F-BV711 (BD), anti-CD64-BV605 (Biolegend), anti-CD24-PE (Biolegend), anti-Ly-6G-PerCP-Cy5.5 (Biolegend), anti-CD103-BV510 (Biolegend), anti-CD86-APC (Biolegend), anti-Ly6C (PE dazzle 594) and anti-MHC II BV421 (Biolegend) for 30 min at 4 °C. Total 10 6 gated events per sample were collected using Fortessa flow cytometer (Becton Dickinson, San Diego, CA), and results were analyzed using FlowJo 10.4.2 software.

Trivedi S., Labuz D., Anderson C., Araujo C.V.d., Blair A., Middleton E.A., Tran A., Mulvey M.A., Campbell R.A., Hale J.S., Rondina M.T., & Leung D.T. (2020). Mucosal-associated invariant T (MAIT) cells mediate protective host responses in sepsis.

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