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Ly6c clone hk1

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Ly6C (clone HK1.4) is a mouse monoclonal antibody that recognizes the Ly6C antigen. Ly6C is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on the surface of various mouse leukocyte subsets.

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Lab products found in correlation

Cd11b clone m1 70, by BioLegend (12 mentions) Cd11c clone n418, by BioLegend (11 mentions) Ly6g clone 1a8, by BioLegend (11 mentions) Cd45 clone 30 f11, by BioLegend (7 mentions) F4 80 clone bm8, by BioLegend (6 mentions) Facscanto 2, by BD (6 mentions) Cd4 clone gk1, by BioLegend (5 mentions) Cd64 clone x54 5 7, by BioLegend (5 mentions) Mhcii clone m5 114, by BioLegend (4 mentions) Cd103 clone 2e7, by BioLegend (3 mentions) Lsrfortessa, by BD (3 mentions) Cd45.1 clone a20, by BioLegend (3 mentions) Cd45 clone 30 f11, by Thermo Fisher Scientific (3 mentions) Facsdiva software, by BD (3 mentions) Cd3 clone 17a2, by BioLegend (2 mentions) Cd24 clone m1 69, by BioLegend (2 mentions) Nk1.1 clone pk136, by BioLegend (2 mentions) Cytofix cytoperm, by BD (2 mentions) Mhc 2 clone m5 114, by Thermo Fisher Scientific (2 mentions) Cd11b clone m1 70, by Thermo Fisher Scientific (2 mentions)

26 protocols using ly6c clone hk1

1

Comprehensive Immune Cell Phenotyping

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The cells were washed in FACS buffer (1% BSA, 0.01% sodium azide, 2.5 mM EDTA in sterile PBS; 650 g, 10 min, 4°C) and preincubated for 15 min with anti-CD16/CD32 Fc-block antibody (10 μg/mL; BioLegend, San Diego, CA, 1:50) in FACS buffer followed by a 30 min incubation with fluorescence dye-conjugated, anti-mouse antibodies for SiglecF (clone E50–2440, 1:400), CD11c (clone N418, 1:100), CD11b (clone M1/70, 1:100), Ly6G (clone 1A8, 1:50, clone 1A8, 1:400), Ly6C (clone HK1.4, 1:80/1:600), F4/80 (clone BM8, 1:100), CD62L (clone DREG-56, 1:80), CD66b (clone G10F5, 1:40), and corresponding isotype controls (all from BioLegend). Analysis was done in adherence to guidelines [25 (link)].
Krenzlin V., Schöche J., Walachowski S., Reinhardt C., Radsak M.P, & Bosmann M. (2023). Immunomodulation of neutrophil granulocyte functions by bacterial polyphosphates. European journal of immunology, 53(5), e2250339.
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2

Multiparameter Flow Cytometry Panel

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CD3 clone 17A2 (BioLegend 100237 and 100244), CD4 clone RM4–5 (BioLegend 100545 and 100510), CD4 clone GK13 (BioLegend 100403), CD8 clone 53–6.7 (BioLegend 100734), CD11b clone M1/70 (BioLegend 101257), CD11c clone N418 (BioLegend 117339 and 117338), CD19 clone 6D5 (BioLegend 115522), CD24 clone M1/69 (BioLegend 101822), CD45 clone 30-F11 (BioLegend 103139, 103132, and 103114; eBioscience 56–0451-82), CD45R clone RA3–6B2 (BioLegend 103247, 103246, and 103226), CD69 clone H1.2F3 (eBioscience 25–0691-81), CD90.2 clone 30-H12 (BioLegend 105331), CD103 clone 2E7 (BioLegend 121406 and 121414), F4/80 clone BM8 (BioLegend 123108), Flt3L (R&D Systems AF427), Ly6C clone HK1.4 (BioLegend 128037), Ly6G clone 1A8 (BioLegend 127645), MHC-II clone M5/114.15.2 (BioLegend 707631), NK1.1 clone PK136 (BioLegend 108707, 108720, and 108749), Streptavidin-Brilliant Violet 650 (BioLegend 405231), Streptavidin-APC (eBioscience 17–4317-82). Depleting antibodies: NK1.1 clone PK136 (BioXCell BE0036), and IgG2a isotype control (BioXCell BE0085).
Barry K.C., Hsu J., Broz M.L., Cueto F.J., Binnewies M., Combes A.J., Nelson A.E., Loo K., Kumar R., Rosenblum M.D., Alvarado M.D., Wolf D.M., Bogunovic D., Bhardwaj N., Daud A.I., Ha P.K., Ryan W.R., Pollack J.L., Samad B., Asthana S., Chan V, & Krummel M.F. (2018). A Natural Killer/Dendritic Cell Axis Defines Responsive Tumor Microenvironments in Melanoma. Nature medicine, 24(8), 1178-1191.
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3

Tumor Cell and Immune Cell Profiling

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As described previously35 , subcutaneous tumours with KP1.9 cells were harvested from C57BL/6 flanks 3 weeks after implantation, minced, and shaken at 600 r.p.m. with 0.2 mg ml−1 collagenase type I (Worthington Biochemical Corporation) in RPMI-1640 for 30 min at 37 °C. Digested samples were filtered (70 μm BD Falcon strainer); washed in PBS with 0.5% BSA and 2 mM EDTA; incubated with Fc-block (TruStain fcX anti-mouse CD16/32; clone 93; Biolegend) for 15 min at 4 °C; and labelled with antibodies as indicated for 45 min at 4 °C. Flow cytometry (LSRII, BD Biosciences) labelled tumour cells (CD45− EpCAM+), TAM (CD45+ CD11b+ Ly6C- Lin- CD11c+ F4/80+), lymphocyte-like cells (CD45+ CD11b- Lin+), along with CD45- EpCAM- host-cell populations. Antibodies included EpCAM (clone G8.8; eBioscience); CD45 (clone 30-F11; Biolegend), F4/80 (clone BM8; Biolegend), CD11c (clone N418; Biolegend), Ly6C (clone HK1.4; Biolegend); and CD11b (clone M1/70; BD Biosciences). The lineage (Lin) antibody mix contained anti-CD90.2 (clone 53–2.1), anti-B220 (clone RA3-6B2), anti-NK1.1 (clone PK136), anti-CD49b (clone DX5), anti-Ter119 (cloneTER-119) and anti-Ly6G (clone 1A8) (all BD Biosciences). 7-aminoactinomycin D (7-AAD, Sigma Aldrich) excluded dead cells. VT680 fluorescence was directly assessed using the LSRII flow cytometer, FlowJo v.8.8.7 (Tree Star, Inc.) and MATLAB.
Cuccarese M.F., Dubach J.M., Pfirschke C., Engblom C., Garris C., Miller M.A., Pittet M.J, & Weissleder R. (2017). Heterogeneity of macrophage infiltration and therapeutic response in lung carcinoma revealed by 3D organ imaging. Nature Communications, 8, 14293.
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4

Identifying Inflammatory Monocytes in Virus Infection

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Mesenteric lymph nodes and Peyer’s patches were collected two days post-infection with the indicated viruses and forced through 70 μM cell strainers to create single cell suspensions. Cells were stained with LIVE/DEAD Aqua Stain (Invitrogen) in PBS according to manufacturer’s instructions. Surface antigens were stained with the following antibodies: CD11b (clone M1/70, BioLegend Cat# 101216) and Ly6C (clone HK1.4, BioLegend Cat# 128025) and for blocking with Rat anti-Mouse CD16/CD32 (Mouse BD Fc Block, Clone 2.4G2, BD Cat# 553142). Cells were then stained for intracellular viral proteins; they were fixed and permeabilized using Cytofix/Cytoperm (BD) at room temperature for 10 minutes then washed in Perm/Wash buffer (BD) and incubated with 1:500 rabbit anti-NS1/2 or mouse anti-NS1 in Perm/Wash buffer for 1 hour at room temperature. Cells were then washed again and stained with secondary goat anti-rabbit antibody (Invitrogen, Cat# 550589) at a dilution of 1:500 in Perm/Wash buffer for 1 hour at room temperature. Data was collected on an LSR Fortessa (BD biosciences) and analyzed using FlowJo (TreeStar). Inflammatory monocytes were gated as CD11b+/Ly6Chigh.
Walker F.C., Hassan E., Peterson S.T., Rodgers R., Schriefer L.A., Thompson C.E., Li Y., Kalugotla G., Blum-Johnston C., Lawrence D., McCune B.T., Graziano V.R., Lushniak L., Lee S., Roth A.N., Karst S.M., Nice T.J., Miner J.J., Wilen C.B, & Baldridge M.T. (2021). Norovirus evolution in immunodeficient mice reveals potentiated pathogenicity via a single nucleotide change in the viral capsid. PLoS Pathogens, 17(3), e1009402.
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5

Isolation and Analysis of Midbrain Mononuclear Cells

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Mononuclear cells were isolated 4 weeks post-transduction from ventral midbrains with bilateral AAV injections, according to published protocols [27 (link), 30 (link)]. Briefly, midbrains were digested with 1 mg/mL Collagenase IV (Sigma) and 20 μg/mL DNAse I (Sigma) diluted in RPMI 1640 with 10% heat inactivated fetal bovine serum, 1% L-glutamine (Sigma), and 1% Penicillin-Streptomycin (Sigma). Mononuclear cells were separated out using a 30/70% Percoll gradient, as previously described [30 (link)]. Isolated cells were blocked with anti-Fcy receptor (clone 2.4G2 BD Biosciences) then incubated with fluorescent-conjugated antibodies against CD45 (clone 30-F11, eBioscience), CD11b (clone M1/70, BioLegend), MHCII (M5/114.15.2, BioLegend), Ly6C (clone HK 1.4, BioLegend), CD4 (clone GK1.5, BioLegend), and CD8a (clone 53-6.7, BioLegend). A fixable viability dye was used to distinguish live cells from debris per manufacturer’s instructions (Fixable Near-IR LIVE/DEAD Stain Kit, Invitrogen). Samples were analyzed using an Attune Nxt flow cytometer (Thermo Fisher Scientific) and FlowJo software (Tree Star).
Williams G.P., Schonhoff A.M., Jurkuvenaite A., Thome A.D., Standaert D.G, & Harms A.S. (2018). Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinson’s disease. Journal of Neuroinflammation, 15, 244.
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6

Multiparametric Flow Cytometry of Liver and Spleen

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For flow cytometric analysis, liver and spleen single-cell suspensions were stained using the following antibodies: Cd11c, clone N418 (Biolegend, San Diego, CA, 117339); Ly6c, clone HK1.4 (Biolegend, 128035); MHCII, clone M5/114.15.2 (eBioscience, Waltham, MA, 48-5321-82); Cd45, clone 30-F11 (eBioscience, 56-0451-82); Cd11b, clone M1/70 (eBioscience, 47-0112-82); Cd64, clone X54-5/7.1 (BD Biosciences, Franklin Lakes, NJ, 741024); Ly6g, clone 1A8 (BD Biosciences, 560601); Fc block Cd16/Cd32, clone 2.4G2 (BD Biosciences, 553142); Ly6g, RB6-8c5 (Tonbo, San Diego, CA, 60-5931); Ghost (Tonbo, 13-0870-T100); Flow cytometric data was acquired on the BD LSRII Fortessa X20 and analyzed using FlowJo (Franklin Lakes, NJ).
Alkhani A., Levy C.S., Tsui M., Rosenberg K.A., Polovina K., Mattis A.N., Mack M., Van Dyken S., Wang B.M., Maher J.J, & Nijagal A. (2020). Ly6cLo non-classical monocytes promote resolution of rhesus rotavirus-mediated perinatal hepatic inflammation. Scientific Reports, 10, 7165.
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7

Flow Cytometric Analysis of Mouse Blood

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For the flow cytometry analysis, mouse whole blood was collected in heparin tube (#366667; BD, NJ, USA) after decapitation under anesthesia. RBC lysis buffer was added to lyse RBC from the whole blood according to manufacturer’s protocol (#420301; BioLegend, CA, USA). Following washing with PBS, cells were incubated with flow antibodies, CD11b (clone M1/70, 1:300), Ly6C (clone HK1.4, 1:300) from BioLegend, CA, USA and TLR9 (clone 26C593.2, 1/300) from Novus Biologicals, LLC, CO, USA. Cells were washed with PBS and fixed with fixation buffer (Affymetrix eBioscience). Samples were run in BD FACSAriaII instrument and were analyzed using BD FACSDiva software (BD Biosciences, San Jose, CA). Fluorescence Minus One (FMO) controls were used to accurately identify the positive cell populations for each marker. Specific markers on the cells were reported as the percentage of the number of gated events. Median fluorescence intensity derived from a fluorescence graph was used to study the level of cell surface expression.
Tripathi A., Bartosh A., Whitehead C, & Pillai A. (2023). Activation of cell-free mtDNA-TLR9 signaling mediates chronic stress-induced social behavior deficits. Molecular Psychiatry, 28(9), 3806-3815.
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8

Multicolor Flow Cytometry of Immune Cells

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LL2 and MC38 cells were purchased from ATCC (Rockville, MD). Cells were cultured in vitro in RPMI-1640 media supplemented with 10% fetal bovine serum, 50 units/mL of penicillin/streptomycin, 2 mM of L-glutamine, 1 mM of sodium pyruvate, and 2 nM of non-essential amino acids, and grown at 37°C. For flow cytometric analyses, antibodies including CD11b (clone M1/70, Biolegend), Gr-1 (Clone RB6–8C5, Biolegend), Ly6C (clone HK1.4, Biolegend), Ly6G (Clone 1A8, Biolegend), RAE-1γ (clone CX1, Biolegend), CD4 (clone GK1.5), CD3ε (clone 145–2C11, BD Bioscience), NK1.1 (clone PK136, Biolegend), Foxp3 (clone PCH101, eBioscience), CD8 (53–6.7, Biolegend), IFNg (XMG1.2, Biolegend) and CD45 (30-F11, Biolegend) were used. Live cell gating was performed using Zombie Aqua Fixable Viability Kit (Biolegend). To detect NKG2D-Fc, we used the PE goat anti-mouse antibodies (Multiple Adsorption, BD Bioscience Cat No: 550589).
Feng P.H., Lam B., Tseng S.H., Kung Y.J., Farmer E., Cheng M.A, & Hung C.F. (2020). NKG2D-Fc fusion protein promotes antitumor immunity through the depletion of immunosuppressive cells. Cancer immunology, immunotherapy : CII, 69(10), 2147-2155.
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9

Investigating Endogenous Antigen Uptake in MICB-Expressing Tumor-Primed DCs

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DCs were isolated from Ctrl-vax- and MICB-vax-immunized mice to investigate whether DCs from MICB-vax-immunized mice had enhanced in vivo uptake of endogenous melanoma antigens. MICB expression was induced on B16F10 (MICB-dox) tumour cells 2 d before DC isolation by doxycycline treatment. Axillary and brachial tdLNs and control mesenteric LNs were collected, and single-cell suspensions were stained with Ghost Violet dead cell exclusion dye (Tonbo Biosciences), followed by Fc blocking with anti-CD16/32 (BioLegend) and surface staining with anti-bodies directed against CD11c (clone N418) and IA/E (clone M5/114.15.2) as well as a panel of dump channel markers, including CD3ε (clone 17A2), CD19 (clone 6D5), TCRβ (clone H57-597), NK1.1 (clone PK136), F4/80 (clone BM8) and Ly6c (clone HK1.4) (all from BioLegend). Migratory DCs (defined as CD3−TCRβ−CD19−NK1.1−Ly6c−F4/80−CD11c+IA/Ehl) were sorted from the stained samples using a FACSAria IIIu instrument (BD Biosciences). CD8+ T cells were isolated from the spleens of naive pmel-1 TCR-transgenic mice, labelled with CTV and co-cultured with sorted migratory DCs at a 5:1 ratio for 72 h. T cell proliferation was measured on the basis of CTV dilution using an LSRFortessa X-20 instrument.
La Teana A., Brandi A., Falconi M., Spurio R., Pon C.L, & Gualerzi C.O. (1991). Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS.. Proceedings of the National Academy of Sciences of the United States of America, 88(23), 10907-10911.
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10

Immune Cell Characterization by Flow Cytometry

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Cells were stained with CD45.2 (clone 104), CD19 (clone 6D5), CD3 (clone 17A2), CD11b (clone M1/70), CD115 (clone AFS98), Ly-6G (clone 1A8), and Ly-6C (clone HK1.4) (all BioLegend). Leukocytes were identified as CD45 high. Myeloid cells were identified as CD45 high CD19 low CD3 low CD11b high. Neutrophils were identified as CD45 high CD19 low CD3 low CD11b high CD115 low Ly-6G high. Inflammatory monocytes were identified as CD45 high CD19 low CD3 low CD11b high Ly-6G low CD115 high Ly-6C high. B-Lymphocytes were identified as CD45 high CD19 high CD3 low.
Seung H., Wrobel J., Wadle C., Bühler T., Chiang D., Rettkowski J., Cabezas-Wallscheid N., Hechler B., Stachon P., Maier A., Weber C., Wolf D., Duerschmied D., Idzko M., Bode C., von zur Mühlen C., Hilgendorf I, & Heidt T. (2022). P2Y12-dependent activation of hematopoietic stem and progenitor cells promotes emergency hematopoiesis after myocardial infarction. Basic Research in Cardiology, 117(1), 16.
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