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Anti ly6c clone hk1

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Anti-Ly6C (clone HK1.4) is a monoclonal antibody that recognizes the Ly6C antigen. Ly6C is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein expressed on various immune cell types, including monocytes, granulocytes, and a subset of T cells.

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Lab products found in correlation

Anti ly6g clone 1a8, by BioLegend (14 mentions) Anti cd11b clone m1 70, by BD (9 mentions) Anti cd11b clone m1 70, by BioLegend (7 mentions) Anti f4 80 clone bm8, by BioLegend (5 mentions) Anti cd45 clone 30 f11, by BD (4 mentions) Anti cd11c clone n418, by BioLegend (4 mentions) Anti cd45 clone 30 f11, by BioLegend (3 mentions) Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (3 mentions) Lsrfortessa, by BD (3 mentions) Anti cd45 clone 30 f11, by Thermo Fisher Scientific (3 mentions) Anti cd25 clone pc61, by BioLegend (3 mentions) Anti cd11c clone hl3, by BD (3 mentions) Anti cd4 clone rm4 5, by BioLegend (3 mentions) Lsr 2, by BD (3 mentions) Sp6800 spectral analyzer, by Sony (2 mentions) Sp6800, by FlowJo (2 mentions) Anti cd64 clone x54 5 7, by BD (2 mentions) Anti b220 clone ra3 6b2, by BioLegend (2 mentions) Anti cd62l clone mel 14, by BioLegend (2 mentions) Anti cd24 clone m1 69, by BD (2 mentions)

Spelling variants of this product

Ly6C (HK1.4, (34 citations) Anti-Ly6C (33 citations) Ly6C (clone HK1.4, (26 citations) Anti-Ly6C (HK1.4, (13 citations) Clone HK1.4 (5 citations) Anti-mouse Ly6C (clone HK1.4, (4 citations) Anti-Ly6C FITC (clone HK1.4; (3 citations) PeCy7-conjugated anti-Ly6C (clone HK1.4), (2 citations) Brilliant Violet 785-conjugated anti-LY6C (clone HK1.4, (2 citations)

23 protocols using anti ly6c clone hk1

1

Flow Cytometric Immune Cell Profiling

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Tumors were collected and processed as above for scRNA-seq and stained with flow cytometry antibodies for 20 min at 4°C. Cells were washed once with PBS and fixed with 1% formalin in PBS before analysis, which was performed on a Sony Biotechnology SP6800 Spectral Analyzer and analyzed with the Sony Biotechnology SP6800 Software and FlowJo (Tree Star, Ashland, OR). Flow cytometry antibodies used in this study were purchased from BioLegend: anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD4 (clone GK1.5), anti-CD45 (clone 30-F11), anti-CD8 (clone 53-6.7), anti-Gr1 (clone RB6-8C5), and anti-Ly6C (clone HK1.4).
Walsh M.J., Ali L.R., Lenehan P., Kureshi C.T., Kureshi R., Dougan M., Knipe D.M, & Dougan S.K. (2023). Blockade of innate inflammatory cytokines TNFα, IL-1β, or IL-6 overcomes virotherapy-induced cancer equilibrium to promote tumor regression. Immunotherapy Advances, 3(1), ltad011.
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2

Brain Tissue Dissociation and Cell Isolation

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Blood was prepared by ACK lysis. Single cell suspensions of brain were prepared as previously described (17 (link), 38 (link)). Mice were euthanized with CO2 and transcardially perfused with ice-cold HBSS. Brains were grossly free of blood. Whole brain was minced, and either mechanically triturated (in experiments for determination of relative proportions of infiltrating cell types) or enzymatically homogenized (in cell sorting experiments) with Neural Tissue Dissociation Kit with Papain, Miltenyi) and passed through a 70 μm cell strainer to form a single cell suspension. Cells were enriched by centrifugation over a discontinuous gradient of HBSS, 37% and 70% Percoll and collecting the fraction at the 37%/70% boundary. Cells were then washed and stained with fluorophore conjugated antibodies prior to analysis on a FACSAria II flow cytometer and cell sorter (BD). Antibodies include anti-CD11b (clone M1/70, BD), anti-CD45 (clone 30-F11, BD), anti-CD64 (clone X54-5/7.1, BD), anti-Ly6G (clone 1A8, Biolegend), anti-Ly6C (clone HK1.4, Biolegend). Propidium iodide was used as a live/dead marker. Gating was performed as described in Figure S1.
Denstaedt S.J., Spencer-Segal J.L., Newstead M.W., Laborc K., Zhao A.P., Hjelmaas A., Zeng X., Akil H., Standiford T.J, & Singer B.H. (2018). S100A8/A9 drives neuroinflammatory priming and protects against anxiety-like behavior after sepsis. Journal of immunology (Baltimore, Md. : 1950), 200(9), 3188-3200.
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3

Comprehensive Immune Profiling of Tumor Microenvironment

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Tumors enzymatically digested by ± 2.3 Wunsch units / ml Liberase TL (Roche, Basel, Switzerland) and tumor draining lymph nodes were passed through 70 μm cell strainers. The generated single cell suspensions were stained with the live/dead marker Zombie Aqua (Biolegend, San Diego, CA, USA) and subsequently blocked for unspecific binding to CD16/32 (TruStain fcX, Biolegend). In order to investigate different myeloid cell populations and endothelial cell activation the following antibodies were diluted in FACS buffer (1% FCS, 0.02% NaN3 and 3 mM EDTA in PBS): anti-CD11b (clone M1/70, Biolegend), anti-CD11c (clone N418, Biolegend), anti-B220 (clone RA3-6B2, Biolegend), anti-CD86 (clone GL-1, Biolegend), anti-CD45 (clone 30-F11, Biolegend), anti-Gr1 (clone RB6-8CS, Biolegend), anti-Ly6C (clone HK1.4, Biolegend), Ly6G (clone 1A8, Biolegend), ICAM-1 (clone YN1/1.7.4, Biolegend), VCAM-1 (clone MVCAM.A, Biolegend) and CD31 (clone MEC 13.3, BD Biosciences, Franklin Lakes, NJ, USA). Samples were washed with FACS-buffer and analyzed in a FACSCanto II cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data analysis was performed with FlowJo software (TreeStar, Ashland, OR, USA).
van Hooren L., Georganaki M., Huang H., Mangsbo S.M, & Dimberg A. (2016). Sunitinib enhances the antitumor responses of agonistic CD40-antibody by reducing MDSCs and synergistically improving endothelial activation and T-cell recruitment. Oncotarget, 7(31), 50277-50289.
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4

Single-cell isolation from brain and spleen

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Brain single cell suspensions were prepared as previously described (17 ). Perfused spleens were dissected, minced and gently macerated through 40 μm cell strainer to form a single cell suspension. Strainers were washed with 30 mL of PBS with 1% bovine serum albumin, 2 mM EDTA, 25 mM HEPES. Cells were washed, Fc receptors blocked, and stained with fluorophore conjugated antibodies prior to analysis on a FACSAria II flow cytometer and cell sorter (BD). Antibodies included anti-CD11b (clone M1/70, BD), anti-CD45 (clone 30-F11, BD), anti-Ly6G (clone 1A8, Biolegend), anti-Ly6C (clone HK1.4, Biolegend). Gating strategy for myeloid cells in the brain and spleen available in supplemental data (Figure S1).
Denstaedt S.J., Spencer-Segal J.L., Newstead M., Laborc K., Zeng X., Standiford T.J, & Singer B.H. (2020). Persistent neuroinflammation and brain specific immune priming in a novel survival model of murine pneumosepsis. Shock (Augusta, Ga.), 54(1), 78-86.
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5

Flow Cytometric Analysis of Immune Cells

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Single cell suspensions from blood, spleen, or tumor were prepared for flow cytometric analysis. Red blood cells in blood were lysed using ACK Lysis Buffer (Life Technologies). Cells were incubated with anti-Fc receptor antibody (clone 2.4G2, BD Biosciences) in Phosphate Buffered Saline (PBS) with 2% fetal bovine serum for 30 mins. Then, surface staining using the following monoclonal antibodies was performed: anti-CD8 (clone 53–6.7, BD Biosciences), anti-CD45 (clone 30-F11, Invitrogen), anti-CD90.1 (clone Ox-7, Biolegend), anti-CD62L (clone MEL-14, Biolegend), anti-CD25 (clone PC61, Biolegend), anti-Ly6C (clone HK1.4, Biolegend), anti-CD11b (clone M1/70, BD Biosciences), anti-CD11c (clone HL3, BD Biosciences), anti-I-Ab (clone AF6–120.1, Biolegend), anti-CD24 (clone M1/69, BD Biosciences), and anti-F4/80 (clone BM8, Biolegend). DAPI or LIVE/DEAD Fixable Near-IR Dead Cell Stain kit (Thermo Fisher)-stained cells were excluded from analysis. Samples were analyzed using LSR II (BD Biosciences) or LSRFortessa (BD Biosciences) with FlowJo software (TreeStar).
Oba T., Hoki T., Yamauchi T., Keler T., Marsh H.C., Cao X, & Ito F. (2020). A critical role of CD40 and CD70 signaling in cDC1s in expansion and antitumor efficacy of adoptively transferred tumor-specific T cells. Journal of immunology (Baltimore, Md. : 1950), 205(7), 1867-1877.
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6

Murine Peritoneal Cell Profiling

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Mice were euthanized with CO2 as described above. Peritoneal exudate was taken from four CLP + Vehicle and five CLP + RvE1 mice. Peritoneal cells were differentiated using anti-CD11b (clone M1/70; eBioscience), anti-F4/80 (clone BM8; BioLegend), anti-Ly6G (clone 1A8; BioLegend), anti-I-A/I-E (clone M5/114.15.2; BioLegend), anti-Ly6C (clone HK1.4; BioLegend), anti-CD64 (clone X54-5/7.1; BioLegend) antibodies. Zombie NIR™ Fixable Viability Kit (Biolegend) was used to identify live cells. Cell counts were determined using Precision Count Beads (Biolegend). Ten thousand live cell events were acquired with a FACSCalibur (Becton Dickinson) and analyzed using FlowJo analysis software (version 9.2, Treestar Inc.).
Chen J., Purvis G.S., Collotta D., Al Zoubi S., Sugimoto M.A., Cacace A., Martin L., Colas R.A., Collino M., Dalli J, & Thiemermann C. (2020). RvE1 Attenuates Polymicrobial Sepsis-Induced Cardiac Dysfunction and Enhances Bacterial Clearance. Frontiers in Immunology, 11, 2080.
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7

Flow Cytometry Antibody Panel for Immune Cell Profiling

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Antibodies to following antigens were used for flow cytometry: anti-CD4 (clone RM4-5, Biolegend #100536), anti-CD5 (clone 53-7-3, Biolegend #100627, #100625), anti-CD8α (clone 53-6.7, Biolegend #100738), anti-CD24 (clone M1/69, Biolegend #101806), anti-CD25 (clone PC61, Biolegend #102016 and #102036), anti-CD44 (clone IM7, Biolegend #103049), anti-CD45.1 (clone A20, Biolegend #110723), anti-CD45.2 (clone 104, Biolegend #109808), anti-CD49d (clone R1-2, Biolegend #103618), anti-CD127 (clone A7R34, Biolegend #135012), anti-TCRβ (clone H57-597, Biolegend #109218), anti-KLRG1 (clone 2F1, Biolegend #138421), anti-Ly6C (clone HK1.4, Biolegend #128006), biotin-conjugated anti-BST2 (clone 927, Biolegend #127006), anti-biotin (clone 1D4-C5, Biolegend #409004). Viability was stained with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen #L34975), Zombie Green™ Fixable Viability Kit (Biolegend, #423111) or Hoechst 33258 (Invitrogen, #H3569). For the analysis of Jurkat cell lines, anti-CD8 (clone MEM-31, Exbio #1P-207-T025), anti-CD69 (clone FN50, Exbio #T7-552-T100) were used. Antibodies were conjugated with various fluorophores and used according to the manufacturer’s instructions.
Paprckova D., Niederlova V., Moudra A., Drobek A., Pribikova M., Janusova S., Schober K., Neuwirth A., Michalik J., Huranova M., Horkova V., Cesnekova M., Simova M., Prochazka J., Balounova J., Busch D.H., Sedlacek R., Schwarzer M, & Stepanek O. (2022). Self-reactivity of CD8 T-cell clones determines their differentiation status rather than their responsiveness in infections. Frontiers in Immunology, 13, 1009198.
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8

Isolation and Characterization of Mouse Myeloid Cells

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Mouse spleen cells were washed with PBS with 1% BSA (staining buffer), blocked with mouse IgG in staining buffer, then stained with conjugated antibodies. Antibodies used were anti-CD11b (clone M1/70, Biolegend, San Diego, CA), anti-Ly-6C (clone HK1.4, Biolegend), and anti-Ly-6G (clone 1A8, Biolegend). Cells were enriched using a mouse CD11b positive selection kit (StemCell Technologies, Vancouver, BC, Canada), then sorted in the BU Flow Cytometry Core.
Muhammad F., Trivett A., Wang D, & Lee D.J. (2019). Tissue-specific production of MicroRNA-155 inhibits melanocortin 5 receptor-dependent suppressor macrophages to promote experimental autoimmune uveitis. European journal of immunology, 49(11), 2074-2082.
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9

Isolation and Analysis of Brain Leukocytes

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Single cell suspensions were prepared as previously described [35 (link)]. Briefly, mice were deeply anesthetized with CO2 and transcardially perfused with ice-cold HBSS. Brains were grossly free of blood after perfusion. Brains were minced, mechanically triturated and passed through a 70 μm cell strainer to form a single cell suspension. Leukocytes were enriched by centrifugation over a discontinuous gradient of 30%, 37%, and 70% Percoll and collecting the fraction at the 37%/70% boundary. Cells were then washed and stained with fluorophore conjugated antibodies as indicated in the text and figures, followed by fixation in paraformaldehyde prior to analysis on an FACSAria II flow cytometer. Antibodies include anti-CCR2 (clone 475301, R&D Systems), anti-CD11b (clone M1/70, BD), anti-CD45 (clone 30-F11, BD), anti-CD64 (clone X54-5/7.1), anti-CX3CR1 (goat anti-mouse polyclonal, R&D Systems), anti-Ly6G (clone 1A8, Biolegend), and anti-Ly6C (clone HK1.4, Biolegend).
Singer B.H., Newstead M.W., Zeng X., Cooke C.L., Thompson R.C., Singer K., Ghantasala R., Parent J.M., Murphy G.G., Iwashyna T.J, & Standiford T.J. (2016). Cecal Ligation and Puncture Results in Long-Term Central Nervous System Myeloid Inflammation. PLoS ONE, 11(2), e0149136.
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10

Immunofluorescence Analysis of Mast Cell Proteases

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Mast cell protease antibodies (Rabbit anti-mouse mMCP-2, dilution: 1/200; Sheep anti-mMCP2, dilution: 1/200; Rabbit anti-mouse mMCP5, dilution: 1/100; Rabbit anti-mouse mMCP6, dilution: 1/500) were a gift from Dr. Michael Gurish. Antibodies to ß-catenin (clone 14, dilution: 1/200,) was purchased from BD Biosciences. CD3ε (clone eBio500A2, dilution: 1/100), and GATA3 (clone TWAJ, dilution: 1/50), were purchased from ebiosciences-Thermo. Alexafluor conjugated (anti-rat, anti-rabbit, anti-sheep, dilution: 1/200) were obtained from Invitrogen-Thermo. For flow cytometry antibodies, anti-CD11b (clone M1/70, dilution: 1/700), anti-CD11c (clone N418, dilution: 1/100), anti-Ly6C (clone HK1.4, dilution: 1/300), anti-Ly6G (clone 1A8, dilution: 1/300), anti-CD117 (clone 2B8, dilution: 1/200), and antiFcεR1 (clone MAR-1, dilution: 1/100) antibodies were purchased from Biolegend. Anti-RORγt (clone Q31-378, dilution: 1/100) was purchased from BD. Anti-Foxp3 (clone FJK-16s, dilution: 1/100) was purchased from ebiosciences-Thermo.
Saadalla A., Lima M.M., Tsai F., Osman A., Singh M.P., Linden D.R., Dennis K.L., Haeryfar S.M., Gurish M.F., Gounari F, & Khazaie K. (2019). Cell Intrinsic Deregulated ß-Catenin Signaling Promotes Expansion of Bone Marrow Derived Connective Tissue Type Mast Cells, Systemic Inflammation, and Colon Cancer. Frontiers in Immunology, 10, 2777.
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