MDSCs were generated as previously reported (35 (link)). Briefly, bone marrow nucleated cells were cultured in DMEM supplied with 2mM L-glutamine, 1.5g/L sodium bicarbonate, 10mM HEPES, 1.0mM sodium pyruvate (all from Gibco, MA, USA), 10% fetal bovine serum (Hyclone, MA, USA), 40ng/ml rmGM-CSF and 40ng/ml rmIL-6 (BioLegend), with or without 50% E.G7-OVA supernatant. The floating cells were collected on day 4 and enriched by anti-mouse Gr-1 Microbeads (Miltenyi). The cells with purity > 90% were used. In some experiments, the cells were stained with anti-mouse Ly6C, anti-mouse Ly6G, and anti-mouse/human CD11b (Biolegend) and sorted with BD FACS Aria II cell sorter (BD).
Anti mouse ly6c
Manufactured by BioLegend
Anti-mouse Ly6C is a cell surface marker that is expressed on a subset of mouse myeloid cells, including monocytes, macrophages, and granulocytes. It is a useful tool for the identification and isolation of these cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse ly6g, by BioLegend (6 mentions)
Anti mouse cd45, by BioLegend (3 mentions)
Anti mouse cd3, by BioLegend (2 mentions)
Trustain fcx, by BioLegend (2 mentions)
Rbc lysis buffer, by BioLegend (2 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Rmgm csf, by BioLegend (1 mentions)
Rmil 6, by BioLegend (1 mentions)
Anti mouse gr 1 microbeads, by Miltenyi Biotec (1 mentions)
Facs symphony, by BD (1 mentions)
Anti mouse f4 80, by BioLegend (1 mentions)
Anti mouse cd11c, by BioLegend (1 mentions)
Il 1beta elisa kit, by Thermo Fisher Scientific (1 mentions)
Trichrome stain masson kit, by Merck Group (1 mentions)
Tgf β1 elisa kit, by Thermo Fisher Scientific (1 mentions)
Cholesterol quantitation kit, by Merck Group (1 mentions)
Mcpt 1 mmcp 1 elisa kit, by Thermo Fisher Scientific (1 mentions)
Anti mouse tnf α, by BioLegend (1 mentions)
6 protocols using anti mouse ly6c
1
Isolation and Characterization of MDSCs
2
Immune Cell Profiling in Tooth Extraction
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The tissue around the tooth sockets and peripheral blood were collected during the inflammatory phase of tissue 1 week after tooth extraction. Peripheral blood was processed by lysing the red blood cells. The tissue was digestion, centrifugation and filtration. Finally, the single cell suspension was stained for immune cell analysis. The cells were incubated with purified anti‐mouse CD45, anti‐mouse F4/80, anti‐mouse CD11b, anti‐mouse CD11c, anti‐mouse Ly6C, anti‐mouse Ly6G and anti‐mouse CD3 (Biolegend) at 4 °C for 30 min. Stained cells were tested on FACS Symphony (BD Biosciences), and data were analysed with FlowJo software.
3
Isolation and Characterization of Murine Myeloid Cells
4
Hepatic Fibrosis Quantification Protocol
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The HFD was purchased from Harlan Laboratories, Inc (Dublin, VA). Oil‐Red‐O staining kit was from Newcomer Supply (Middleton, WI). Trichrome Stain (Masson) Kit and Cholesterol Quantitation Kit were from Sigma‐Aldrich (Louis, MO). Triglyceride Quantification Colorimetric/Fluorometric Kit and Alanine Aminotransferase (ALT or SGPT) Activity Colorimetric/Fluorometric Assay Kit were from BioVision Incorporated (Milpitas, CA). Mouse LIPA/Lysosomal Acid Lipase Sandwich ELISA Kit was purchased from LSBio Lifespan BioSciences, Inc (Seattle, WA). Mouse tumor necrosis factor‐α (TNF‐α) ELISA Kit, interleukin (IL)–6 ELISA, IL‐1 beta ELISA Kit, MCPT‐1 (mMCP‐1) ELISA Kit, and human/mouse transforming growth factor‐β (TGF‐β) 1 ELISA Kit were purchased from eBioscience (San Diego, CA). Anti‐mouse alpha smooth muscle actin (MSC), anti‐mouse F4/80 and anti‐SQSTM1/62 antibodies were from Abcam (Cambridge, MA). The anti‐mouse Ly6G, anti‐mouse Ly6C, anti‐mouse CD11b, anti‐mouse C‐X‐C motif chemokine receptor 2, and anti‐mouse TNF‐α were from BioLegend (San Diego, CA). Anti‐mouse IL12p40 and anti‐mouse Gr‐1 antibody was from eBioscience (San Diego, CA).
5
Immune Cell Profiling by Flow Cytometry
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Cells were subjected to fluorescence-activated cell sorting (FACS) analysis with specific antibodies. The antibodies used for cell surface staining were: anti-mouse CD45 and anti-mouse CD206 (TONBO); anti-mouse CD11b, anti-mouse CD11c, anti-mouse CD8, anti-mouse F4/80, and anti-mouse CD209a (eBioscience); anti-mouse CD4, anti-mouse CD3, anti-mouse Ly6C, and anti-mouse Ly6G (Biolegend). Data were acquired with an LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo V10.6 (Tree Star Inc.).
6
Isolation and Characterization of Murine Myeloid Cells
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Bone marrow was harvested from femurs and red blood cells were lysed (RBC lysis buffer, Biolegend). Bone marrow cells were blocked with TruStain FcX (Clone 93, Biolegend) and stained with anti-mouse CD45.2 (Clone 104, Biolegend) or IgG2a κ Isotype (Clone MOPC-173, Biolegend), anti-mouse CD11b (Clone M1/70, Biolegend) or IgG2b, κ Isotype (Clone RTK4530, Biolegend), anti-mouse Ly6G (Clone 1A8, Biolegend) or IgG2a, κ Isotype (Clone RTK2758, Biolegend) and anti-mouse Ly6C (Clone HK1.4, Biolegend) or IgG2c, κ Isotype (Clone RTK4174, Biolegend). Samples were analysed using a BD FACS Canto II and FlowJo software.
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