Anti ly6c

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-Ly6C is a laboratory reagent used for the detection and identification of Ly6C, a glycosylphosphatidylinositol (GPI)-anchored protein expressed on the surface of various mouse cell types. It is commonly used in flow cytometry and other immunological applications to study the distribution and function of Ly6C-positive cells.

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PE-conjugated anti-Ly6C antibody (HK1.4, (4 citations) Anti-Ly6C (clone HK1.4, (4 citations) Anti-Ly6c (HK1.4, (4 citations) Anti-Ly6C eFluor 450 (3 citations) Anti-mouse Ly6C (2 citations) Rat anti-mouse Ly6C FITC (2 citations) PE-conjugated anti-mouse Ly6C (2 citations) Anti-Ly6C (AL-21; (2 citations) Anti-Ly6C-PE Cy5.5 (2 citations) Anti-mouse Ly6C-PE (2 citations)

23 protocols using anti ly6c

Comprehensive Flow Cytometry Analysis of Immune Cells

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Hepatic and adipose immune cells were pre‐incubated with anti‐mouse CD16/32 Fc blocker (BD Pharmingen, USA), followed by staining with the Live/Dead marker anti‐FVD‐APC‐Cy7 (all supplied by eBioscience, San Diego, CA, USA). The fluorochrome‐conjugated antibodies used in this study were anti‐CD45, anti‐CD3, anti‐NK1.1, anti‐CD4, anti‐CD8, anti‐CD44, anti‐CD62L, anti‐CD11b, anti‐F4/80, anti‐Ly6C, anti‐Ly6C, and anti‐Siglec‐F (all supplied by eBioscience, San Diego, CA, USA). Liver mononuclear cells were stimulated with phorbol‐myristate acetate/ionomycin/brefeldin A/monensin for 5 hr in vitro. The cells were fixed and permeabilized using a Fixation/Permeabilization Buffer kit (eBioscience, San Diego, CA, USA). The permeabilized cells were washed with FACS buffer and resuspended in 1% formaldehyde and stained for intracellular cytokines with anti‐IFN‐γ‐PE‐Cy7, anti‐TNF‐α‐APC, and anti‐IL‐17A‐APC fluorochrome‐conjugated antibodies. Stained cells were analyzed using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA), and data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, USA).

Moon J.S., Goeminne L.J., Kim J.T., Tian J.W., Kim S., Nga H.T., Kang S.G., Kang B.E., Byun J., Lee Y., Jeon J., Shong M., Auwerx J., Ryu D, & Yi H. (2020). Growth differentiation factor 15 protects against the aging‐mediated systemic inflammatory response in humans and mice. Aging Cell, 19(8), e13195.

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Characterization of Hepatic Tumor Immune Cells

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Hepatic tumorous immune cells were preincubated with anti-mouse CD16/32 Fc blocker (BD Pharmingen, San Diego, CA, USA), followed by staining with the Live/Dead marker FVD-APC-Cy7 (all supplied by eBioscience, San Diego, CA, USA). The fluorochrome-conjugated antibodies used in this study were anti-CD45, anti-CD3, anti-NK1.1, anti-CD4, anti-CD8, anti-CD44, anti-CD62L, anti-CD279, anti-CD11b, anti-F4/80, anti-Ly6C, anti-Ly6C, anti‐Siglec-F, anti-MCL-1, and anti-BCL2 (all supplied by eBioscience). Hepatic tumor immune cells were stimulated with phorbol-myristate acetate/ionomycin/brefeldin A/monensin for 5 hours in vitro. Cells were fixed and permeabilized using the Fixation/Permeabilization Buffer kit (eBioscience), washed with FACS buffer, resuspended in 1% formaldehyde, and stained with anti‐IFN-γ-APC. Stained cells were analyzed using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and the data were analyzed using the FlowJo software (FlowJo, LLC, Ashland, OR, USA). The antibodies used are listed in online supplemental table S3.

Song B.S., Moon J.S., Tian J., Lee H.Y., Sim B.C., Kim S.H., Kang S.G., Kim J.T., Nga H.T., Benfeitas R., Kim Y., Park S., Wolfe R.R., Eun H.S., Shong M., Lee S., Kim I.Y, & Yi H.S. (2022). Mitoribosomal defects aggravate liver cancer via aberrant glycolytic flux and T cell exhaustion. Journal for Immunotherapy of Cancer, 10(5), e004337.

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Flow Cytometry Analysis of Murine Immune Cells

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Spleens were homogenized by manual disruption and red cells were lysed as described above. The cells were resuspended at 4°C in FACS buffer (PBS, 5% FBS and 0.1% sodium azide). Antibodies were incubated with cells for 30 min at 4°C at the indicated dilution and washed with FACS buffer before resuspension in fresh FACS buffer. Samples were analyzed on a Cyan flow cytometer (Dako) or an LSR-II flow cytometer (BD). The antibodies used were as follows: anti-Ly6G (48–5931-82; eBioscience; dilution 1/200), anti-CD11c (17–0114-81; eBioscience; dilution 1/200), anti-CD19 (48–0193-82; eBioscience; 1/200), anti CD11b (17–0112-82 or 45–0112-80; eBioscience), anti-CD8a (11–0081-82; eBioscience; 1/400 dilution), anti-CD4 (558107; BD), anti-F4/80 (12–4801-82; eBioscience; 1/200), anti-CD115 (12–1152-82; eBioscience; 1/250), anti CD45.1 (25–0453-82; eBioscience; 1/200), anti-CD45.2 (17–0454-81; eBioscience; 1/200), and anti-Ly6C (53–5932; eBioscience; 1/200).

Thomas D.C., Clare S., Sowerby J.M., Pardo M., Juss J.K., Goulding D.A., van der Weyden L., Storisteanu D., Prakash A., Espéli M., Flint S., Lee J.C., Hoenderdos K., Kane L., Harcourt K., Mukhopadhyay S., Umrania Y., Antrobus R., Nathan J.A., Adams D.J., Bateman A., Choudhary J.S., Lyons P.A., Condliffe A.M., Chilvers E.R., Dougan G, & Smith K.G. (2017). Eros is a novel transmembrane protein that controls the phagocyte respiratory burst and is essential for innate immunity. The Journal of Experimental Medicine, 214(4), 1111-1128.

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Multiparametric Tissue Analysis Protocol

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Histologic analysis was performed on snap frozen tissues that were fixed in acetone, blocked with 10% FCS, and stained with anti-active caspase-3 (BD Biosciences), anti-cleaved caspase-8 (Cell Signaling Technology), anti-PD-L1, anti-LY6C, anti-LY6G (all eBioscience), and anti-BAFF (R&D) antibodies. Fluorescent images were taken with an Axiocam 503 color microscope (ZEISS) and quantified using ImageJ. Conventional histology images were taken using the Brightfield microscope.

Liu W., Stachura P., Xu H.C., Váraljai R., Shinde P., Ganesh N.U., Mack M., Van Lierop A., Huang A., Sundaram B., Lang K.S., Picard D., Fischer U., Remke M., Homey B., Roesch A., Häussinger D., Lang P.A., Borkhardt A, & Pandyra A.A. (2021). BAFF Attenuates Immunosuppressive Monocytes in the Melanoma Tumor Microenvironment. Cancer Research, 82(2), 264-277.

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Antibody Panel for Microglial Analysis

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The following antibodies were used for this study: P2ry12 (1:500), a generous gift from Dr. Oleg Butovsky; CD68 (1:200), Abcam ab53444, RRID:AB_869007; 4G8 (20 μg/reaction), Biolegend^® RRID:AB_10175149; Anti‐CD11b (1:100), BD Biosciences Cat# 552850 RRID:AB_394491; anti‐CD45 (1:200), eBioscience Cat# 17‐0451‐82 162 RRID:AB_469392; anti‐Ly6C (1:400), eBioscience Cat# 12‐5932‐82 RRID:AB_10804510; anti‐Ly6G (1:100), Biolegend 163 Cat# 127605 RRID: AB_1236488; GAPDH (1:5,000), Sigma‐Aldrich G9545 RRID: AB 796208; β2AR (1:500), Abcam ab182136 (lot: GR302897‐20) RRID: AB not available.
Detailed methods regarding environmental enrichment setup, mouse brain tissue staining and microglial morphology analysis, microglia isolation and RNA analysis, and human brain extract preparation and analysis are reported by previous publications (Yang et al, 2013, 2017; Xu et al, 2016). Methods unique to this study are listed below:

Xu H., Rajsombath M.M., Weikop P, & Selkoe D.J. (2018). Enriched environment enhances β‐adrenergic signaling to prevent microglia inflammation by amyloid‐β. EMBO Molecular Medicine, 10(9), e8931.

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Immunostaining of Lung Tissue Sections

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Immediately following euthanasia, left lung lobes were dissected and immersion fixed in 1% paraformaldehyde-lysine-periodate fixative (1% paraformaldehyde in 0.2M lysine-HCL, 0.1M anhydrous dibasic sodium phosphate, with 0.21% sodium periodate, pH 7.4) for 24 hours at 4°C. Following fixation, lungs were placed in a 30% w\v sucrose solution for 24 hours at 4°C, prior to embedding and freezing in O.C.T. compound (Tissue Tek). Embedded tissues were sectioned at 5μm for immunostaining. Nonspecific binding was blocked by pre-incubation of sections with 5% donkey serum (Jackson ImmunoResearch, West Grove, PA) in 1% BSA for 30 min at RT. Primary antibody labeling (1:200 anti-GFP, Novus Biologicals; 1:100 anti-F4/80 clone BM8, eBiosciences, and 1:100 anti-Ly6C ab76975) was performed at RT for 1hr in 1% BSA. After removal of the primary antibody, tissues were washed with PBS-T, followed by addition of the following secondary antibodies (diluted 1:200 in PBST) for 30 min at RT: AlexFluor488-conjugated donkey anti-rabbit IgG (GFP), AlexaFluor647-cojugated donkey anti-rat IgG (F4/80), and Cy3-conjugated donkey anti-rat IgG (Ly6C) (Jackson ImmunoResearch). Tissues were counter stained with DAPI, cover-slipped, and visualized using an Olympus IX83 confocal microscope and Hamamatsu digital camera. Figures were assembled using Adobe Photoshop (CC2016).

Regan D.P., Coy J.W., Chahal K.K., Chow L., Kurihara J.N., Guth A.M., Kufareva I, & Dow S.W. (2019). The Angiotensin Receptor Blocker Losartan Suppresses Growth of Pulmonary Metastases via AT1R-Independent Inhibition of CCR2 Signaling and Monocyte Recruitment. Journal of immunology (Baltimore, Md. : 1950), 202(10), 3087-3102.

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Comprehensive Immune Cell Profiling

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Isolated cells from spleens, joint footpads, and pLN were first stained with a Live/Dead fixable aqua dead cell staining kit (1:400) (Life Technologies) for 20 min. Cell were then washed and resuspended in 50 μl blocking buffer (1% rat and hamster serum in PBS). Staining was performed using anti-CD45 (BD Pharmingen), anti-CD4 (BD Pharmingen), anti-CD8 (eBioscience), anti-CD25 (eBioscience), anti-CD11b (eBioscience), anti-Ly6C (eBioscience), and anti-Ly6G (BioLegend) antibodies for 20 min. Stained cells were then washed with PBS and fixed using IC fixation buffer (eBioscience). Intracellular staining of anti-Foxp3 (eBioscience) was done according to the manufacturer's instructions. Data acquisition and analyses were done as described above.

Lee W.W., Teo T.H., Her Z., Lum F.M., Kam Y.W., Haase D., Rénia L., Rötzschke O, & Ng L.F. (2015). Expanding Regulatory T Cells Alleviates Chikungunya Virus-Induced Pathology in Mice. Journal of Virology, 89(15), 7893-7904.

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Isolation and Characterization of Mouse Monocytes

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Bone marrow cells were isolated by flushing femurs of mice with RPMI-1640 containing 2% FBS, and spleens were homogenized through 40 uM nylon mesh. Resulting cell suspensions were pelleted by centrifugation at 300x g. RBCs were lysed and washed three times and counted. Absolute cell numbers were calculated based on the percentage of monocytes from the total cell population acquired by flow cytometry. Single-cell suspensions were blocked with CD16/CD32 (2.4G2) and then stained variously with CD4 (RM4–5), CD8a (53–6.7), B220 (RA3–6B2), NK1.1 (PK136), CD11b (M1/70), Ly6C (HK1.4), Ly6G (1A8), Siglec-F (E50–2440). Live/Dead fixable dead cell stain kit (Invitrogen) was used to remove dead cells. Monocytes were gated as Ly6C^hiCD11b⁺ from non-CD4, -CD8, -B220, -NK1.1, -Ly6G and -Siglec-F. All samples were acquired using a FACS LSRII (BD Biosciences) and analyzed using FlowJo software (Tree Star). All antibodies were purchased from BD Biosciences except anti-CD4 and anti-Ly6C (eBioscience).

Jeong Y.H., Walsh M.C., Yu J., Shen H., Wherry E.J, & Choi Y. (2020). Mice lacking the purinergic receptor P2X5 exhibit defective inflammasome activation and early susceptibility to Listeria monocytogenes. Journal of immunology (Baltimore, Md. : 1950), 205(3), 760-766.

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Quantifying Immune Cell Phenotypes in Mice

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To detect CD1d1 on the surface of immune cells, leukocytes from mouse blood were collected. On the other hand, the colonic tissues from indicated mice were isolated and were liberated by digestion with Liberase C (1.6 U/ml; Roche) and deoxyribonuclease I (0.2 mg/ml; Roche) for 2 hours at room temperature. The dead cells were excluded first by staining with the LIVE/DEAD Fixable Near-Infrared Dead Cell Stain Kit (Life Technologies, Eugene, OR, USA). To measure the expression of phenotype markers on the cell surface, suspended cells were incubated for 1 hour at room temperature in the dark using fluorescent antibodies (anti-CD11c, anti-B220, anti-NK1.1, anti-CD4, anti-CD8, anti-CD45, anti-Foxp3, anti-Ly6G, anti-Ly6C, and anti-F4/80; eBioscience, San Diego, CA, USA). A total of 10,000 live cells were analyzed by FACsAria cytometer (BD, Franklin Lakes, NJ, USA). The gate strategy is shown in fig. S9. All flow cytometry data were analyzed using CellQuest Pro software.

Cui S., Wang C., Bai W., Li J., Pan Y., Huang X., Yang H., Feng Z., Xiang Q., Fei L., Zheng L., Huang J., Zhang Q., Wu Y, & Chen Y. (2020). CD1d1 intrinsic signaling in macrophages controls NLRP3 inflammasome expression during inflammation. Science Advances, 6(43), eaaz7290.

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Intracellular Cytokine Staining of Immune Cells

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For intracellular-cytokine staining, single-cell suspended splenocytes were incubated with brefeldin A (eBioscience), followed by an additional 5 h of incubation at 37°C. After surface staining with anti-CD3, anti-CD8, anti-CD11b, anti-CD11c, anti-CD19, anti-CD115, anti-F4/80, anti-Ly6C, anti-Ly6G, anti-MHC-II, and anti-NK1.1 antibodies (all from eBioscience), the cells were fixed with 2% formalin, permeabilized with 0.1% saponin, and stained with anti-TNF antibodies (eBioscience) for 30 min at 4°C. B-cell subsets were detected in single-cell suspensions of splenocytes with anti-CD5, anti-CD19, anti-CD21, anti-CD23, and anti-IgM antibodies (all from eBioscience). BD Calibrite (BD Biosciences, USA) beads were added to the samples before acquisition with a BD LSRFortessa.

Shinde P.V., Xu H.C., Maney S.K., Kloetgen A., Namineni S., Zhuang Y., Honke N., Shaabani N., Bellora N., Doerrenberg M., Trilling M., Pozdeev V.I., van Rooijen N., Scheu S., Pfeffer K., Crocker P.R., Tanaka M., Duggimpudi S., Knolle P., Heikenwalder M., Ruland J., Mak T.W., Brenner D., Pandyra A.A., Hoell J.I., Borkhardt A., Häussinger D., Lang K.S, & Lang P.A. (2018). Tumor Necrosis Factor-Mediated Survival of CD169+ Cells Promotes Immune Activation during Vesicular Stomatitis Virus Infection. Journal of Virology, 92(3), e01637-17.

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