Lungs cells were harvested from mice at 3 days p.i. and processed as previously described by us (18 (link), 19 (link), 22 (link)). Cells types in the lungs were quantified by staining with Pacific Blue™ anti-mouse CD11b (Clone M1/70), APC-Cy7 anti-mouse CD11c (Clone N418), FITC anti-mouse CD19 (Clone 6D5), Brilliant Violet 570 anti-mouse CD3 (Clone 17A2), APC anti-mouse Ly6G (Clone 1A8), PerCP/Cyc5.5 anti-mouse Ly6C (Clone HK1.4), PE-Cy7 anti-mouse F4-80 (Clone BM8) antibodies (Biolegend, San Diego, CA), and PE anti-mouse TCR β (Clone H57-597) antibody (Becton Dickinson Pharmingen, San Jose, CA). Enumeration of neutrophils by flow cytometry (using a BD LSR II, Becton Dickinson, San Jose, CA) was done by quantitating Ly6G+CD11b+ cells stained with Pacific Blue™ anti-mouse CD11b (Clone M1/70) and APC anti-mouse Ly6G (Clone 1A8) antibodies (Biolegend, San Diego, CA). FlowJo (Tree Star) software was used to analyze all data.
Apc anti mouse ly6g
Manufactured by BioLegend
Sourced in United States
The APC anti-mouse Ly6G is a flow cytometry antibody that binds to the Ly6G antigen expressed on mouse neutrophils. It can be used to identify and quantify neutrophils in mouse samples.
Automatically generated - may contain errors
Lab products found in correlation
Pe anti mouse cd11b, by BioLegend (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rpe anti mouse cd31, by BD (2 mentions)
Osteosense, by PerkinElmer (2 mentions)
F3506, by Merck Group (2 mentions)
Atc2000, by World Precision Instruments (2 mentions)
Formyl methionyl leucyl phenylalanine (fmlp), by Merck Group (2 mentions)
Tween 20, by Thermo Fisher Scientific (2 mentions)
Electrophoresis solution, by Beyotime (2 mentions)
Transfer solution, by Beyotime (2 mentions)
Fitc anti mouse cd4, by BioLegend (2 mentions)
Apc anti mouse cd25, by BioLegend (2 mentions)
Anti mouse percp cy5.5 ly6c, by BioLegend (2 mentions)
Tbs buffer, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Beyotime (2 mentions)
Anti mouse fitc gr1, by BioLegend (2 mentions)
Dmem medium, by Thermo Fisher Scientific (2 mentions)
Cck 8 kit, by Beyotime (2 mentions)
Paraformaldehyde (pfa), by Beyotime (2 mentions)
Rt pcr primers, by Sangon (2 mentions)
13 protocols using apc anti mouse ly6g
1
Quantification of Lung Cell Types
2
Multicolor Flow Cytometry of Murine Blood
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For flow cytometry, murine blood was diluted in Tyrode’s buffer and washed twice via centrifugation at 650 g for 5 min. Afterwards, the centrifuged supernatant was removed and only the cell pellet was used for further analysis. The blood samples were incubated with antibodies for leucocytes (adenomatous polyposis coli protein [APC] anti-mouse CD45; BD-Bioscience), neutrophils (APC anti-mouse Ly6G, Biolegend [San Diego, CA, USA]) and platelets (PE anti-mouse glycoprotein [GP]Ib, EMFRET Analytics) for 15 min at room temperature. The reaction was stopped by adding PBS and the samples were analysed immediately on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Only the mean fluorescence intensity of double-positive cells, indicating platelet/leucocyte or platelet–neutrophil aggregates, was evaluated.
3
Poly(I:C)-Induced Inflammation Modulation
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Poly(I:C) was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Dexamethasone (DXM) was purchased from Solarbio Biotechnology Co. Ltd. (Beijing, China). Total protein, superoxide dismutase (SOD), methane dicarboxylic aldehyde (MDA), glutathione peroxidase (GSH-Px), inducible nitric oxide synthase (iNOS), and myeloperoxidase (MPO) assay kits were obtained from the Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). Enzyme-linked immunosorbent assay (ELISA) kits of mouse arginase 1 (Arg-1), and interleukin (IL)-1β, IL-6, IL-10, and IL-4 were purchased from Multi Science Biotechnology Co. Ltd. (Hangzhou, China). ELISA kits of mouse tumor necrosis factor-alpha (TNF-α) were purchased from Shanghai BlueGene Biotech Co., Ltd. (Shanghai, China). PE anti-mouse CD86, APC anti-mouse CD206, APC anti-mouse Ly6G, and PE anti-mouse CD11b antibodies were purchased from BioLegend, Inc. (Beijing, China).
4
Visualizing Skull Channel Cell Traffic
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To explore cell traffic through the skull channels to the meninges, we employed an organ bath system. After in vivo staining of the bone surface with OsteoSense® 750EX (4 nmol/mouse, PerkinElmer) the day before the experiment, the vasculature and neutrophils were stained with IV injections of 50 µl of RPE anti mouse CD31 (#553373 BD Biosciences) and 50 µl of APC anti mouse Ly6G (#127614 Biolegends) antibodies 1 hour before harvest. Using surgical scissors, we obtained a skull piece of about 4 by 4 mm, respecting the integrity of the dura and the visible edges of the marrow cavities. This specimen was flipped upside down and rapidly transferred into the organ bath. The specimen was placed on a stage and immersed with a solution of 50 µmolar fMLP (F3506 Sigma-Aldrich), used as chemoattractant, in HBSS. This protocol was adopted from previously published in vitro neutrophil migration assays35 (link). The organ bath was maintained at 37°C through a warming plate (ATC-2000 World Precision Instruments). The same conditions were implemented to explore neutrophil migration through the channels at 4–6 hours after permanent MCAO or after intracisternal injection of carrageenan, and corresponding sham or naive controls. Neutrophils exiting the channels were identified and counted using Imaris software™ (Bitplane).
5
Immunoblot Analysis of NLRP3 Inflammasome
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Anti-β-actin (1:10,000, BH10D10) was bought from Cell Signaling Technology (Danvers, MA, United States); Anti-NLRP3 (1:1,000. Cryo-2) and Anti-ASC (1:1,000, AL177) were purchased from Adipogen (San Diego, CA, United States); Anti-Caspase-1 (1:1,000, ab179515) and Anti-NEK7 (1:10,000 ab133514) were bought from Abcam (Cambridge, CB2 0AX, United Kingdom); Anti-IL-1β (1:000 AF-401-NA; RRID: AB_416684) was obtained from RD systems (Tustin, CA, United States); the DyLight 488-labeled secondary antibody (1:50, A120-100D2) was purchased from InvivoGen (San Diego, CA, United States); and FITC anti-mouse/human CD11b (101216, 1:500 for flow cytometry) and APC anti-mouse Ly-6G (127614, 1:500 for flow cytometry) were from BioLegend.
6
Visualizing Skull Channel Cell Traffic
7
Cellular Characterization of Murine Peritonitis
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Exudate cells from the murine peritonitis model were prepared to determine the cellular composition. The cells were blocked with mouse anti-CD16/CD32 (BioLegend, #101320, 1:50) antibodies for 10 min at room temperature and then stained with anti-mouse APC-Ly6G (BioLegend, # 127614, 1:250), anti-mouse e450-F4/80 (eBioscience, # 48-4801-82, 1:100) (and anti-mouse FITC-Ly6C (BioLegend, #128006, 1:250) antibodies for 30 min at 4 °C. To analyze the MΦ phagocytosis of apoptotic PMNs in vivo, the cells were permeabilized and then stained with anti-mouse PerCP-Cy5.5-conjugated anti-Ly6G (BioLegend, #127616, 15:1000) for 30 min at 4 °C. The cells were analyzed by flow cytometry (BD FACSCanto II). All FACS gating strategies are shown in Supplementary Fig. 9 . Cytokines, RGM-A and Norepinephrine were measured in the murine peritoneal exudates using standard ELISA (R&D systems, LDN immunoassays).
8
Tumor and Spleen Cell Isolation Protocol
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The DMEM medium, PBS medium, and 10% fetal bovine serum were purchased from Gibco Life Technologies (Grand Island, NY, USA). The tumor dissociation kit and spleen dissociation kit were obtained from Miltenyi Biotec (San Diego, CA, USA). Anti-mouse PerCP/Cy5.5 CD3, anti-mouse FITC CD4, anti-mouse PE CD8, anti-mouse APC CD25, anti-mouse FITC Gr-1, anti-mouse PE CD11b, anti-mouse PerCP/Cy5.5 Ly6c, and anti-mouse APC Ly6g were all purchased from Biolegend (San Diego, CA, USA). 5%BSA, paraformaldehyde, electrophoresis solution, transfer solution, CCK-8 kit, RIPA were all provided by Beyotime Biotech (Beijing, China). The SDS-PAGs (Sodium Dodecyl Sulfate-Polyacrylamide Gels) were bought from Dakewe Biotech (Shenzhen, China). RT-PCR primers were designed and provided by Sangon Biotech (Shanghai, China). Trizol reagent, Tween-20, and 20 × TBS buffer were obtained from Thermo Scientific (Rockford, IL). Antibodies for Western blot were all purchased from Cell Signaling Technology (MA, USA). An ECL Kit was provided by Tanon Biotech (Shanghai, China).
9
Analyzing Gemcitabine's Apoptotic Effects
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The DMEM medium and 10% FBS were obtained from Gibco Life Technologies (Grand Island, NY, USA). Gemcitabine (GEM) was brought from Selleckchem. The PBS medium, Annexin V-FITC/PI apoptosis detection kit, and BCA protein assay kit were purchased from KeyGEN Biotech (Jiangsu, China). 5%BSA, paraformaldehyde (PFA), electrophoresis solution, transfer solution, CCK-8 kit, RIPA, and SDS-PAGE gel were all provided by Beyotime Biotech (Beijing, China). The mouse tumor and spleen dissociation kits were obtained from Miltenyi (MACS, Miltenyi Biotec, Germany). Anti-mouse PE-CD11b, anti-mouse FITC-Gr-1, anti-mouse PerCP/Cy5.5-Ly6C, anti-mouse APC-Ly6G, anti-mouse PE NKG2D, anti-mouse APC-CD49b, anti-mouse FITC-CD4, and anti-mouse APC-CD25 were purchased from BioLegend. RT-PCR primers were designed and provided by Sangon Biotech (Shanghai, China). Trizol reagent, Tween-20, and 20× TBS buffer were obtained from Thermo Scientific (Rockford, IL). An ECL Kit was provided by Tanon Biotech (Shanghai, China).
10
Granulocyte Colony-Stimulating Factor Treatment in Mice
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B6.SJL-PtprcaPepcb/BoyCrl mice (Ly5.1 mice) were maintained under pathogen-free conditions in the research animal facility of the University of Tübingen, according to German federal and state regulations (Regierungspräsidium Tübingen, M 05-20G). Mice, aged between 6 and 8 weeks, were treated with intraperitoneal injections (i.p.) of rhG-CSF, Boskar4, Boskar4_t2, or Boskar4_st2 at a concentration of 300 µg/kg every second day for a total of five injections. Mice were sacrificed one day after the last injection. Mice in the control group were treated with PBS, or Moevan_control using the same schema. Bone marrow cells were isolated by flushing with a 22 G syringe, and filtered through a 0.45 µm cell strainer prior to counting and staining for flow cytometry analyses. Cells were stained with DAPI (Sigma#D9542), anti-mouse CD45.1 PerCP (Biolegend#110725), anti-mouse Ly6C AF488 (Biolegend#128022), and anti-mouse Ly6G APC (Biolegend#127614). Flow cytometry was carried out on a FACS Canto II (BD), and data were analysed using FlowJo (BD). Vital mononuclear cells were selected, and doublets were excluded based on scatter characteristics. Gates were set according to fluorescence minus one (FMO) controls.
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