The colonic lamina propria cells were blocked with rat serum then stained with live/dead dye FVS700 (BD Biosciences) and the fluorophore-conjugated surface marker antibodies BV605-conjugated anti-CD45 (BD Biosciences), anti-CD11B, and anti-Ly6G antibody (Biolegend). After surface staining, cells was fixed and permeabilized with Foxp3/transcription factor staining buffer set (Thermo Fisher Scientific, MA, USA) followed by staining with AF647-anti S100A9 (BD Biosciences). Cells was acquired on BD FACSCanto™ II (BD Biosciences) and data were analyzed with FlowJo® ver. 10 (BD Biosciences).
Anti ly6g antibody
Manufactured by BioLegend
Sourced in United States
The Anti-Ly6G antibody is a laboratory reagent used for the detection and identification of Ly6G, a cell surface marker expressed on neutrophils and a subset of myeloid-derived suppressor cells (MDSCs). This antibody can be used in various immunological techniques, such as flow cytometry, to analyze the presence and distribution of Ly6G-positive cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b antibody, by BioLegend (2 mentions)
Fvs700, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Anti cd11b, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Isotype igg, by Cell Signaling Technology (1 mentions)
70 μm strainer, by Thermo Fisher Scientific (1 mentions)
Automated cell counter, by Thermo Fisher Scientific (1 mentions)
Streptavidin beads, by STEMCELL (1 mentions)
Dextran coated magnetic particles, by STEMCELL (1 mentions)
Anti cd19, by BioLegend (1 mentions)
Anti cd4, by BioLegend (1 mentions)
Anti cd8, by BioLegend (1 mentions)
Modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Igepal co 520, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Zinc nitrate hexahydrate zn no3 2 6h2o, by Merck Group (1 mentions)
Pkh67, by Thermo Fisher Scientific (1 mentions)
Anti cd206 antibody, by BioLegend (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
18 protocols using anti ly6g antibody
1
Colonic Immune Cell Profiling
2
Neutrophil Depletion and NSA Administration in Murine Myocardial Infarction
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Details for reagents and antibodies described in the Methods can be found in Supplemental Table 2 .
Adult C57BL/6N mice were purchased from Shanghai SLAC Laboratory. C57BL/6N Gsdmd−/− mice were purchased from GemPharmatech. All animals were housed in a pathogen-free environment in the Tongji University animal facilities. Knockout of the Gsdmd gene was validated by genotyping and immunoblotting. All animal surgeries were performed in 10- to 14-week-old male mice.
To deplete neutrophils in mice, isotype IgG (Cell Signaling Technology), anti-Ly6G antibody (BioLegend), or anti-Ly6G/Ly6C antibody (BioLegend) was injected intraperitoneally at a dose of 200 μg per mouse, as described previously (30 (link), 31 (link)). The relevant mice were subjected to myocardial infarction surgery 24 hours later. For NSA administration, 20 mg/kg mouse weight of NSA (MedChemExpress) was injected intraperitoneally 30 minutes before AMI surgery; a second injection was administered 8 hours after the surgery. Mice in the corresponding control groups were injected with the same amount of solvent (DMSO) without NSA.
Adult C57BL/6N mice were purchased from Shanghai SLAC Laboratory. C57BL/6N Gsdmd−/− mice were purchased from GemPharmatech. All animals were housed in a pathogen-free environment in the Tongji University animal facilities. Knockout of the Gsdmd gene was validated by genotyping and immunoblotting. All animal surgeries were performed in 10- to 14-week-old male mice.
To deplete neutrophils in mice, isotype IgG (Cell Signaling Technology), anti-Ly6G antibody (BioLegend), or anti-Ly6G/Ly6C antibody (BioLegend) was injected intraperitoneally at a dose of 200 μg per mouse, as described previously (30 (link), 31 (link)). The relevant mice were subjected to myocardial infarction surgery 24 hours later. For NSA administration, 20 mg/kg mouse weight of NSA (MedChemExpress) was injected intraperitoneally 30 minutes before AMI surgery; a second injection was administered 8 hours after the surgery. Mice in the corresponding control groups were injected with the same amount of solvent (DMSO) without NSA.
3
Quantifying Neutrophil-Candida Interaction
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2.5 × 104 murine bone marrow neutrophils were incubated for 1 h at 37°C with either opsonized or non-opsonized pACT1-GFP C. albicans yeast cells at a fungus:neutrophil ratio of 1:1. After washing, neutrophils were incubated with anti-Ly6G antibody (BioLegend) for 20 min at 4°C. The degree of C. albicans uptake was determined by counting the number of GFP+ neutrophils relative to the total number of neutrophils by flow cytometry.
4
Isolation of Neutrophils from Ire1α-deficient Mice
5
Isolation of Murine Organ Neutrophils
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Neutrophils from lymphoid organs (spleen and lymph nodes) were isolated by first removing T, B, and DC populations using biotin-labeled anti-CD4 (#100404, Biolegend), anti-CD8 (#100704, Biolegend), anti-CD19 (#115504, Biolegend), and anti-CD11c antibodies (#117304, Biolegend) with streptavidin beads (#19860, Stemcell Technologies), followed by neutrophil positive selection using a biotin-labeled anti-Ly6G antibody (#127604, Biolegend) and dextran-coated magnetic particles (#18556, Stemcell Technologies). Isolated neutrophils were used for reactive oxygen species (ROS) quantitation, co-culture, and qPCR studies.
6
Neutrophil Depletion and Subarachnoid Hemorrhage
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To elucidate the effect of neutrophil depletion treatment on EBI, 72 mice were randomly divided into three groups (n = 24/group): sham, SAH + vehicle (saline), and SAH + anti-Ly6G antibody. For neutrophil depletion, anti-Ly6G antibody (1A8 BioLegend 127,649) was intravenously injected into mice at a dose of 5 µg/g mouse at 24 h before SAH [25 (link), 26 (link)]. SAH severity, neurological scores (beam balance and modified Garcia tests), brain water content, Evans blue dye extravasation, immunofluorescence staining, and western blotting were evaluated at 24 h after SAH induction.
7
DOPA-PtdSer Nanoparticles for Arthritis
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Dioleoylphosphatydic acid (DOPA) and dioleoyl phosphatidylserine (PtdSer) were purchased from A.V.T. (Shanghai) Pharmaceutical Co., Ltd. (Shanghai, China). Metformin, cyclohexane, Igepal CO-520 and zinc nitrate hexahydrate (Zn(NO3)2·6(H2O)) were obtained from SigmaAldrich (St. Louis, MO, USA). Low-molecular-weight-heparin (LMWH, MW 3800~5000, Mw/Mn (PDI) = 1.34) was purchased from Melonepharma (Dalian, China). Recombinant murine macrophage colony-stimulating factor (M-CSF) (315-02) and recombinant human TNF (300-01 A) were purchased from PeproTech (Rocky Hill, USA). RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), modified Eagle’s medium, trypsin EDTA, type I collagenase, fetal bovine serum (FBS), and PBS were obtained from Gibco (USA). DiR (D12731) and PKH67 were purchased from Invitrogen (USA). The anti-CD206 antibody, anti-iNOS antibody, anti-Ly-6G antibody, and anti-myeloperoxidase (MPO) antibody were purchased from BioLegend. All siRNAs used in the CIA model were synthesized by Genepharma (Shanghai, China), and the sequences used were as follows: (i) IRF5: 5-dTdT-CUG CAG AGA AUA ACC CUG A-dTdT-3 (sense) and 5-dTdT UCA GGG UUA UUC UCU GCA G dTdT-3 (antisense). A dye was introduced at the 5′-end of the antisense strand of siIRF5. (ii) Negative control scrambled siRNA (siN.C).
8
Neutrophil Isolation and Immunoblotting
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Bone marrow cells were flushed from one femur and one tibia per mouse with RPMI media (ThermoFisher, catalog no. 61870044) supplemented by 10% FBS. Red blood cells were lysed with ACK (Ammonium-Chloride-Potassium) buffer46 (link). Neutrophils were magnetically isolated using anti-Ly6G antibody (Clone: 1A8, BioLegend) according to manufacturer’s instructions from Miltenyi Biotec. Purity was checked by Attune™ NxT Flow Cytometer. The cells were lysed with RIPA Buffer (Pierce, Thermo Scientific), and then subjected to electrophoresis and transferred to nitrocellulose membranes (ThermoFisher, catalog no. LC2001, Sweden). Membranes were incubated with antibodies for NCF1 (Clone: D-10, Santa Cruz Biotechnology) and cyclophilin A (ThermoFisher, catalog no. PA1-025, Sweden) at 1:1000 dilution, followed by incubation with HRP-conjugated goat anti-rabbit IgG (ThermoFisher, catalog no. 31460) at 1:20,000 dilution. The ChemiDoc XRS + Gel imaging system (Bio-Rad Laboratories AB, Sweden) was used for chemiluminescence detection.
9
Isolation and Flow Cytometry of Cochlear Immune Cells
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After both sides of the temporal bones were isolated from the mice, the bony capsule of the cochlea was carefully removed in PBS. The whole cochlea tissue was extracted from the cochlear bony capsule and temporal bone. After the cochlear tissue was transferred to a new dish, the tissue was trypsinized for 10 min, followed by gentle grinding on a 40 μm filter. The cells were stained with dye-conjugated anti-CD11b antibody (Biolegend, 1:200) and anti-Ly6G antibody (Biolegend, 1:200) for 30 min at 4 °C. Flow cytometry was performed using BD LSR II (Becton Dickinson, Franklin Lakes, NJ, USA). The analysis was performed using the FlowJo software (Becton Dickinson).
10
Neutrophil Isolation and Flow Cytometry
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Cells were isolated from the spleen 24 h after surgery using the Mouse Kidney Dissociation Kit (Miltenyi Biotec) in accordance with the manufacturer’s instructions; cells were also isolated from the bone marrow by rinsing with sterile PBS. The proportion of neutrophils was then analyzed using flow cytometry (ACEA NovoCyte) with the anti-Ly6G antibody (Biolegend), anti-Gr-1 antibody (Biolegend), and Zombie (Biolegend) for 30 min at 4 °C. All flow cytometry data were analyzed using FlowJo software (Tree Star).
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