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).
Percp cy5.5 anti ly6c
Manufactured by BioLegend
PerCP/Cy5.5-anti-Ly6C is a fluorochrome-conjugated antibody that binds to the Ly6C antigen expressed on the surface of certain immune cells. It can be used for the identification and analysis of Ly6C-positive cell populations in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti ly6g fitc, by BioLegend (2 mentions)
Acridine orange, by Merck Group (1 mentions)
Anti cd31 pe cy7, by BioLegend (1 mentions)
Anti cd11b apc, by Thermo Fisher Scientific (1 mentions)
Goat anti clec4f, by R&D Systems (1 mentions)
Streptavidin bv605, by BioLegend (1 mentions)
Pe anti ep cam, by BioLegend (1 mentions)
Anti cd11b pe, by BioLegend (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Facs canto flow cytometer, by Tree Star (1 mentions)
Anti cd11b fitc, by BioLegend (1 mentions)
Apc cy7 conjugated streptavidin, by BioLegend (1 mentions)
7 aad, by BD (1 mentions)
3 protocols using percp cy5.5 anti ly6c
1
Isolated Liver Immune Cell Profiling
2
Single-Cell Surface Marker Profiling
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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
Comprehensive Phenotypic Characterization of Blood Cells
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BM cells were suspended in FACS buffer (2.5% BSA; 0.5 mM EDTA, in PBS) following red cell-lysis with 0.8% NH4Cl buffer. Phenotypic surface markers were labeled using PE-conjugated anti-CD3/B220/Ter119, FITC anti-CD11b, PerCP-Cy5.5 anti-Ly6C, APC anti-Ly6G or CD11b, and Biotin anti-CD115 antibodies and APC Cy7 conjugated streptavidin (Biolegend). Cells were stained with 7AAD (BD Bioscience, UK) to assess cell death. For hematopoietic stem cell (HSC) analysis, PE-conjugated lineage cocktail and PE-conjugated isotype antibodies were used alongside FITC anti-Sca-1 and APC anti-CD117 antibodies. Data were acquired using a FACS Canto flow cytometer and analyzed using FlowJo Software (Tree Star Inc, OR, USA, version 8.8.7) and populations were gated using isotype and fluorescence minus one controls.
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