Isolated liver immune cells and cultured liver macrophages were collected and counted in an automated cell counters (Cellometer Auto X4, Nexelcom Bioscience) after staining for acridine orange (Sigma-aldrich). Antibodies (details in table S2 ) including BUV396-anti-CD45 (BD bioscience; # 563791), FITC-anti-Ly6G (Biolegend; #127605), APC/Cy7-anti-F4/80 (Biolegend; #123117), PerCP/Cy5.5-anti-Ly6C (Biolegend; #128011), PE/Cy7-anti-CD31 (Biolegend; #102417), Alexa488-anti-iNOS (Thermo Fisher; #53-5920-82), APC-anti-CD11b (Thermo Fisher; 17-0112-82), PE-anti-Tim4 (Thermo Fisher; 12-5866-82), or goat anti-Clec4f (R&D systems; #AF2784) were incubated with FACS buffer (2% fetal bovine serum, 2 mM EDTA, and sodium azide in PBS) on ice for 30 min. In some experiments, the primary cultures of KCs were incubated with 100 ng/ml of biotinylated LPS (Invivogen, #tlrl-lpsbiot) for 2 hours. Then biotin was detected using PE/Cy7-streptividin (Biolegend; #405206). After surface staining of biotin, in some experiments, internalized biotin-LPS was stained using BV605-streptavidin (Biolegend; #405229) in cells permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (Thermo eBioscience; #88-8824). After washing and resuspension, cells were analyzed on a BD FACS Symphony machine and analyzed by FlowJo software (BD biosciences).
Anti ly6g fitc
Manufactured by BioLegend
Sourced in United States
Anti-Ly6G-FITC is a fluorescently-labeled monoclonal antibody that binds to the Ly6G antigen. Ly6G is a glycosylphosphatidylinositol-anchored protein expressed on the surface of mature neutrophils. This product can be used for the identification and enumeration of neutrophils in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
7 aad, by BioLegend (3 mentions)
Anti cd11b pe cy7, by BioLegend (3 mentions)
Dnase, by Merck Group (3 mentions)
Anti ly6c apc cy7, by BioLegend (3 mentions)
Anti f4 80 pe, by BioLegend (3 mentions)
Percp cy5.5 anti ly6c, by BioLegend (2 mentions)
Anti cd11b apc cy7, by BioLegend (2 mentions)
Anti cd3 fitc, by BioLegend (2 mentions)
Anti cd45 efluor450, by Thermo Fisher Scientific (2 mentions)
Anti f4 80 pe, by Thermo Fisher Scientific (2 mentions)
Anti cd3 a700, by Thermo Fisher Scientific (2 mentions)
Anti cd8 brilliantviolet605, by BioLegend (2 mentions)
Anti cd11b pe cy5, by Thermo Fisher Scientific (2 mentions)
Anti cd64 apc, by BioLegend (2 mentions)
Anti cd4 pe cy7, by BioLegend (2 mentions)
Collagenase d, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
70 μm cell strainer, by Corning (2 mentions)
Anti cd45 percp cy5, by BioLegend (2 mentions)
16 protocols using anti ly6g fitc
1
Isolated Liver Immune Cell Profiling
2
Single-Cell Surface Marker Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For surface staining, single-cell suspensions were incubated with FACS antibodies in FACS buffer, which consisted of PBS with 2% BSA and 5 mM EDTA, for 20 min at 4 °C. The antibodies used for staining included APC/Cy7 anti-CD45.2 (Biolegend, clone 104), PE anti-Ep-CAM (Biolegend, clone G8.8), PE anti-CD11b (Biolegend, clone M1/70), FITC anti-Ly6G (Biolegend, clone 1A8), PerCP/Cy5.5 anti-Ly6C (Biolegend, clone HK1.4). Flow cytometric analyses were performed using a cytometer (CytoFlex s, Beckman Coulter) and the acquired data were analyzed using FlowJo v.10.0.7 software.
3
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometric analysis was performed as described previously, with modifications [24 (link)]. In brief, the cell suspension in 2% newborn calf serum (NCS) PBS was stained with an anti-CD16/32 antibody (TruStain fcX) (Biolegend) to avoid non-specific staining. After being washed with 2% NCS PBS, the cells were further stained with the following antibodies: FITC-anti-Ly6G (Biolegend), FITC-anti-CD63 (gift from Dr. Kurashima, The University of Tokyo) [25 (link)], APC-Cy7-anti-CD11b (Biolegend), APC-anti-FcεRI (eBioscience), PE-anti-c-kit (BD Biosciences), PE-Cy7-anti-CD45 (Biolegend), BV421-anti-CD45 (Biolegend), and BV421-anti-Siglec-F (Biolegend). Dead cells were detected by using 7-AAD (Biolegend) and were excluded from the analysis. Flow cytometry analysis was performed by using MACSQuant (Miltenyi Biotec, Bergisch Gladbach, Germany) or FACSAria (BD Biosciences). Data were analyzed by using FlowJo 9.9 (Tree Star, Ashland, OR, USA).
4
Dissociation and Analysis of Tumor-Derived Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumour masses were harvested from mice, dissociated with a Gentlemacs instrument (Miltenyi), and digested in DMEM medium containing 1 mg/ml collagenase IA (C2674, Sigma) and 0.02 mg/ml DNAse (D4513, Sigma) at 37°C for 30 min (program 37C_m_LPDK), then filtered with 70‐mm cell strainers (Becton and Dickinson), centrifuged and resuspended in PBS. Resuspended cells were counted by cytometry. The cells were stained with fluorescence‐labelled antibodies in buffer (PBS with 1% BSA, 5 mM EDTA, 0.01% NaN3): FITC‐anti‐Ly6G (#127605, Biolegend), PE‐anti‐CD3 (#553064, BD), PE‐Cy7‐anti‐CD11b (#101215, Biolegend), APC‐Fire750‐anti‐Epcam (#118230, Biolegend), V450‐anti‐CD45.2 (#560697, BD), BV650‐anti‐CD45R‐B220 (#103241, Biolegend), PE‐Dazzle594‐anti‐CD31 (#102429, Biolegend) and Zombie Yellow (#423103, Biolegend), acquisition was performed on the SONY ID7000 (SONY) and analysed using Sony software.
5
Comprehensive Immunological Analysis via Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For flow cytometry, we used FITC anti-CD3, PE anti-CD8, APC anti-CD4, APC anti-NK1.1, APC anti-CD11b, FITC Anti-Ly6G, PE anti-IFN-α, APC CD11-C, and FITC anti-F4/80 antibodies (all from BioLegend). The antibodies used for the in vivo neutralization experiments were purchased from BioXcell (anti-mouse IFNAR-1, Clone: MAR1-5A3, Catalog number: BP0241, and mouse IgG1 isotype control, Clone: MOPC-21, Catalog number: BE0083). Anti-Ly6G (Sigma-Aldrich; MABF474), anti-F4/80 (Abcam; ab6640), anti-IFN-α-FITC conjugated (R&D Systems; 22100–3), anti-cleaved caspase-3 (Cell Signaling Technology; 9661S), anti-CD163 (Santa Cruz Biotechnology, INC; sc-58965), anti-Ly6C (Santa Cruz Biotechnology, INC; sc-52650), anti-CD115 (Santa Cruz Biotechnology, INC; sc-46662), anti-CD200 (Santa Cruz Biotechnology, INC; sc-53100), anti-CD11c (Abcam; ab33483), anti-CD68 (Abcam; ab53444), anti-RIP-1/3 (Santa Cruz Biotechnology, INC; sc-133102/sc-374639) and secondary antibodies (goat anti-rat IgG (H+L) -Alexa 647, goat anti-rabbit IgG (H+L), Alexa Fluor 488, and goat anti-mouse IgG (H+L), Alexa Fluor 594) were obtained from Life Technologies, and fluoroshield mounting medium with DAPI (Abcam, ab104139) was used for the confocal microscopy analyses.
6
Urinary Immune Cell Profiling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bladders were digested at 37 °C for 30 min in RPMI-1640 with 10 mM HEPES, collagenase D, and DNAse (all from Sigma-Aldrich), forced through a 70 μm cell strainer (Corning), and washed with 5% fetal bovine serum (FBS, ThermoFisher Scientific, Durham, NC, USA) in PBS. Urines were diluted in fluorescence-activated single-cell sorting (FACS) Buffer (PBS supplemented with 5 mM EDTA and 3% FBS) for 30 min (pH neutralization), prior to staining. Single-cell suspensions resuspended on ice-cold FACS Buffer were stained with anti-CD45 eFluor450 (eBioscience, San Diego, CA, USA), anti-CD3-A700 (eBioscience), anti-CD4-PE/Cy7 (BioLegend, San Diego, CA, USA), anti-CD8-BrilliantViolet605 (BioLegend), anti-CD11b-PE/Cy5 (eBioscience), anti-Ly6C-APC/Cy7 (BioLegend), anti-Ly6G-FITC (BioLegend), anti-F4/80-PE (eBioscience), anti-CD64-APC (BioLegend), and 7-AAD (BioLegend). Purified anti-FcɣRIII/II (BioLegend) was used to block Fc-receptors. Data were acquired on LSR II flow cytometer (BD) and analyzed with FlowJo software v10.0 (BD).
7
Isolation and Analysis of Lung and Liver Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PM2.5 was administered to the mouse under the same conditions as described above. Lungs and livers of mice were chopped for the preparation of single cells. Lung tissue was incubated for 1 h at 37°C, 150 rpm in 5 ml HBSS containing 100 μl of 5% collagenase type IV, 150 μl of 0.1 M CaCl2, and 25 μl of DNase I (1 mg/ml). The digested lung tissue was filtered into 70 μm cell strainer. Single cells were obtained from the lung by centrifuging the filtered solution at 1,500 rpm at 4°C for 3 min. Liver tissue was incubated in 10 ml HBSS containing 500 μl of 5% collagenase type IV and 25 μl of DNase I (1 mg/ml) for 30 min at 37°C, 150 rpm. The digested liver tissue was filtered into a 70 μm cell strainer. Single cells were obtained from the liver by centrifuging the filtered solution at 400 g at 4°C for 10 min. For flow cytometry analysis of single cells from the lung and the liver, T cells were stained with anti-CD3-APC/Cy7 (BioLegend) and macrophages were stained with anti-F4/80-PE (BioLegend) and neutrophils were stained with anti-Ly6G-FITC (BioLegend). Fluorescence was measured using LSRII or LSRFortessa (BD Biosciences). The data were analyzed using FlowJo software.
8
Immune Cell Profiling of Bladder Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three to five bladder tissues of each group were dissociated in Liberase TL (0.5 mg/mL, Merck#05401020001) and dispase II (20 mg/mL, Merck#D4693) in HEPES-buffer saline (sigma) for 120 min at 37 °C. Dissociated tissues were filtered through 70 mm mesh and resuspended in HBSS buffer with 2% PFA. Cells were centrifuged at 500g for 3 min and resuspended in HBSS buffer. Immune cells were separated by 40% and 80% Percoll solution before flow cytometry. Surface antigens were stained and fixated and permeabilized, then stained intracellular proteins. Antibodies used for staining: anti-CD45.2-AF700 (1:200, Biolegend# 109822), anti-CD11b-APC-CY7 (1:200, Biolegend# 101226), anti-Ly-6G-FITC (1:200, Biolegend# 127606), anti-F4/80-PE (1:200, Biolegend# 157304), anti-CD86-FITC (1:200, Biolegend# 105110), anti-CD206-APC (1:200, Biolegend# 141708). We excluded dead cells by using a live cell stain (AquaTM Fixable viability, Biolegend). It was centrifuged at 500g for 3 min and resuspended in HBSS buffer. Flow cytometry was conducted on Gallios flow cytometry (Beckman). Data were analyzed by FlowJo software.
9
Comprehensive Flow Cytometry Analysis of Lung and Lymph Node Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BALF cells recovered were labeled with anti‐CD11c‐APC, anti‐Ly6G‐FITC, anti‐CD11b‐PE‐Cy7 (Biolegend), anti‐CD45‐APC‐Cy7, and anti‐Siglec‐F‐PE (BD Biosciences) for 45 min. MLN cells were isolated. The cell clumps were disaggregated into single‐cell suspensions using nylon mesh (70 μm pore size) filtration, and erythrocytes were lysed with RBC lysis. For detection of Th1, Th2, Th17, and Treg cells, the MLN cells isolated above were stimulated with lymphocyte activator mixture for 5 h and labeled with surface markers anti‐CD4‐V450 (BD Biosciences), anti‐CD4‐FITC (eBioscience), or anti‐CD25‐APC (Biolegend). After washing, fixing, and permeabilizing according to the manufacturer's instructions, cells were labeled intracellularly with anti‐IL‐17A‐PE‐Cy7, anti‐Foxp3‐PE (Biolegend), anti‐IFN‐γ‐APC, and anti‐IL‐13‐PE (Thermo Fisher Scientific), and then were incubated for 45 min. After rinsing, all labeled cells were detected using FCM on the FACScan Flow Analyzer. Data were analyzed with FlowJo software.
10
Multicolor Flow Cytometry Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were blocked with 1 µg anti-CD16/32 per 106 cells for 15 min at 4 °C. Cells were incubated with antibodies in the dark for 1 h on ice. After washing with 1% BSA in PBS, cells were filtered and resuspended in the cell staining buffer for characterization of murine immune cell subsets according to a previously reported protocol [31 ]. The following antibodies were used: anti-CD3-BV650, anti-CD11b-Pacific Blue, anti-CD11c-PE/Cy7, anti-CD19-BV605, anti-CD45-Per-CP-Cy5.5, anti-MHC-II-BV510, anti-F4/80-PE, and anti-Ly6G-FITC (BioLegend, CA, USA). Data acquisition was performed on a CytoFLEX Flow Cytometer (Beckman Coulter, CA, USA) and analyzed with Kaluza software (version 4.2, Beckman Coulter).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!