Ly6g af647

Manufactured by BioLegend

Ly6G-AF647 is a fluorophore-conjugated antibody that specifically binds to the Ly6G antigen, which is a marker for neutrophils. This product can be used for the identification and analysis of neutrophils in flow cytometry applications.

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Anti-Ly6G-AF647 (2 citations) Anti-Ly6G-AF647 (clone 1A8, (2 citations)

5 protocols using ly6g af647

Inducing NETosis in Ceacam 1 Knockout Mice

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Peripheral blood from Ceacam 1 knockout (CC1 KO) and C57 BL/6 mice were isolated by heart puncture and lysed with BD cell lysis buffer as per manufacturer’s instructions (BD Biosciences). Neutrophils were sorted following staining with Ly6G-AF647 (1:200) (1A8; BioLegend, San Diego, CA) and then stimulated with 500 nM PMA for 4 h at 37°C, 5% CO₂ to induce NETosis.

Rayes R.F., Vourtzoumis P., Rjeily M.B., Seth R., Bourdeau F., Giannias B., Berube J., Huang Y.H., Rousseau S., Camilleri-Broet S., Blumberg R.S., Beauchemin N., Najmeh S., Cools-Lartigue J., Spicer J.D, & Ferri L.E. (2020). Neutrophil Extracellular Trap–Associated CEACAM1 as a Putative Therapeutic Target to Prevent Metastatic Progression of Colon Carcinoma. Journal of immunology (Baltimore, Md. : 1950), 204(8), 2285-2294.

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Sepsis Model in BALB/c Mice

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Nine-week-old male BALB/c mice were used for these experiments. Animals were anaesthetized by isoflurane (Forene). After abdominal incision, the cecum was ligated, punctured with a gauge needle (25G), and a small amount of fecal matter was released. For the sham group, after abdominal incision, the cecum was manipulated but was neither ligated nor punctured. After the cecum was returned to the abdomen, the abdominal cavity was closed in two layers and the mice were resuscitated with 30 ml kg⁻¹ body weight of saline (0.9% NaCl) administered subcutaneously. LCC-12 (0.3 mg kg⁻¹, intraperitoneal injection) was administered at 4 h, 24 h, 48 h, 72 h and 96 h following CLP creation. Mortality incidence was monitored every 2 h up to 120 h (except from 10 pm to 6 am) after CLP creation. Dexamethasone was administered intraperitoneally at 1 mg kg⁻¹ 5 min before CLP creation. ICP-MS experiments were conducted on SPMs as described in ‘ICP-MS’. Tissue-specific data were normalized against dry weight. The sorting of SPMs was done using the following antibodies: CD11b-Pacific Blue (BioLegend, 101224), F4/80-PE (TONBO, TNB50-4801-U100), Ly6C–PerCP/Cy5.5 (BioLegend, 128012) and Ly6G–AF647 (BioLegend, 127610). The sorted SPMs corresponded to CD11b⁺F4/80^intLy6C⁻Ly6G⁻ cells.

Solier S., Müller S., Cañeque T., Versini A., Mansart A., Sindikubwabo F., Baron L., Emam L., Gestraud P., Pantoș G.D., Gandon V., Gaillet C., Wu T.D., Dingli F., Loew D., Baulande S., Durand S., Sencio V., Robil C., Trottein F., Péricat D., Näser E., Cougoule C., Meunier E., Bègue A.L., Salmon H., Manel N., Puisieux A., Watson S., Dawson M.A., Servant N., Kroemer G., Annane D, & Rodriguez R. (2023). A druggable copper-signalling pathway that drives inflammation. Nature, 617(7960), 386-394.

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Liver Immune Cell Isolation and FACS Analysis

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Flow cytometry was performed on cells isolated from mice, as described previously [36 (link)]. Liver lobes were cut up and digested in 2.5 mg/ml collagenase (Roche, 11088866001) in 3 % FBS for 45 minutes at 37 °C. Tissues were cut up and mashed through 70 μm nylon filters in 10 ml of PBS. Cells were spun down, then red blood cell lysis was performed using Pharm Lyse (BD Biosciences, 555899) at 4 °C for 5 minutes, then quenched with PBS. For liver, blood cells were separated from the rest of the tissue using a 33 % percoll (GE Life Sciences, 17089102) separation spun at 800 g for 30 minutes. Cells were blocked for 30 minutes in MACS buffer (0.5 % FBS, 200 μM EDTA) and stained with the following antibodies: CD45-FITC (Biolegend, 147710), CD11b-BV605 (BD Horizon, 563015), Ly6G-AF647 (Biolegend, 127610), F4/80-BV711 (BD Horizon, 565612), Ly6C-biotin (Biolegend, 128003), streptavidin-APCefluor780 (eBioscience, 47-4317082), CD22-PE (Biolegend, 126111), CD19-APCCy7 (BD Pharmingen, 557655). T cells were stained using CD44-FITC, CD62L-PE, CD25-PE-Cy7, CD8-APC, and CD4-BV421. Flow cytometry was run on the Attune NxT in the Cytometry and Imaging Microscopy Shared Resource of the Case Comprehensive Cancer Center. Data was analyzed using FlowJo.

Oswald D.M., Zhou J.Y., Jones M.B, & Cobb B.A. (2020). Disruption of Hepatocyte Sialylation Drives a T cell-Dependent Pro-Inflammatory Immune Tone. Glycoconjugate journal, 37(3), 395-407.

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Aorta Cell Staining Protocol

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Staining of cells isolated from aortas was done as described before(35 (link)). Cell suspensions were stained with CD41-FITC (MW Reg30) (BD Pharmingen), Ly6G-AF647 (Biolegend), Ly6C-PerCP-Cy5.5 (HK1.4) (eBiociences) and previously described antibodies (35 (link)).

Koltsova E.K., Sundd P., Zarpellon A., Ouyang H., Mikulski Z., Zampolli A., Ruggeri Z.M, & Ley K. (2014). Genetic deletion of platelet Glycoprotein Ib alpha but not its extracellular domain protects from atherosclerosis. Thrombosis and haemostasis, 112(6), 1252-1263.

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Isolation and Characterization of Immune Cells

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Mice were euthanized by CO₂ exposure, and the organs were harvested and placed on ice. After collecting all groups, the organs were minced into tiny pieces using fine surgical scissors and digested with 1 mg/ml Collagenase IV and 1 mg/ml DNase I at 37°C for 40 min. The digest was then forced through 70 μm cell strainers using a 5 ml syringe plunger. The filtrates were centrifuged at 400 g for 5 min to remove cell debris. After removing the supernatant, the cell pellets were resuspended in 2 ml of 30% Percoll (GE HealthCare) and carefully layered onto 80% Percoll in a 15 ml tube without disturbing the interface. The tube was then centrifuged at 1000 g for 15 min at room temperature. The cells at the interface between the 30% and 80% layers were collected and washed with flow staining buffer (1% BSA with 0.05% sodium azide). Isolated immune cells were counted using a hemocytometer, and 10⁶ cells were collected for further analysis. Prior to staining, cells were blocked with Fc receptor blocking antibody at room temperature for 30 min and stained with the appropriate antibodies. All antibodies (CD45 APC-Cy7, CD11b PE-Cy7, Ly6C PerCP, Ly6G AF647, F480 FITC, NK1.1 PE) were purchased from BioLegend. The cells were analyzed using a FACS Aria III flow cytometer. The resulting FCS file was analyzed using FlowJo V10 software.

Zhu W., Zhang H., Dong Q., Song H, & Zhao L. (2023). Dual wave of neutrophil recruitment determines the outcome of C. albicans infection. Frontiers in Cellular and Infection Microbiology, 13, 1239593.

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