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Anti ly6c pe

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Anti-Ly6C-PE is a fluorescent-labeled antibody used for the detection and analysis of Ly6C, a cell surface antigen, in flow cytometry applications.

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Lab products found in correlation

Anti cd45 apc cy7, by BioLegend (5 mentions) Anti cd11b apc, by BioLegend (5 mentions) Anti cd206 apc, by BioLegend (3 mentions) Anti ly6g apc cy7, by BioLegend (3 mentions) Anti cd4 fitc, by BioLegend (3 mentions) Anti cd11b fitc, by BioLegend (3 mentions) Anti cd138 pe cy7 clone 281 2, by BioLegend (3 mentions) Anti cd11c bv421, by BioLegend (2 mentions) Anti ly6g pe, by BioLegend (2 mentions) Anti f4 80 apc, by BioLegend (2 mentions) Facs fortessa flow cytometer, by BD (2 mentions) Anti cd19 pe cf594, by BD (2 mentions) Anti cd49b pe cf594, by BD (2 mentions) Anti cd45 bv605, by BD (2 mentions) Anti cd11b percp cy5, by BioLegend (2 mentions) Anti cd11c bv650, by BioLegend (2 mentions) Anti f4 80 alexafluor 700, by BioLegend (2 mentions) Anti mhc 2 fitc, by BioLegend (2 mentions) Anti cd45 bv605, by BioLegend (2 mentions) Anti cd3 bv650, by BioLegend (2 mentions)

Spelling variants of this product

Anti-Ly6C (33 citations) Ly6C-PE (14 citations) PE-conjugated anti-Ly6C (3 citations) PE-conjugated anti-mouse Ly6C (3 citations) Anti-Ly6C (PE dazzle 594), (2 citations) PE/Cy7‐conjugated anti‐Ly6C mAb (2 citations) PE/Cy7 anti-Ly6C (2 citations) Anti-mouse Ly6C-PE (2 citations)

18 protocols using anti ly6c pe

1

Kupffer Cell and MDSC Characterization

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Cells were incubated with Fc-blocking reagent (TrueStain, Biolegend) for 5 min, and subsequently stained for 20 min at 4°C with the relevant antibody. Some antibodies were used to analyze Kupffer cells: FITC anti-F4-80 (BM8), APC anti-I-A-I-E (M5/114.15.2), PE anti-CD86 (GL-1), PECy7 anti-CD68 (FA-11), PerCP anti-CD11b (M1/70) (Biolegend), PE anti-CD273 (122), and anti-CD274 (MIH5) (eBiosciences). For experiments involving Kupffer cell/MDSC co-culture, Kupffer cells were first labeled with CFSE (0.5 µM, Invitrogen). MDSC were characterized with the following antibodies: APC anti-CCR2 (475301, R&D Systems), APC anti-CD244.2 (eBio244F4, eBiosciences), APC anti-CD54 (YN1/1.7.4), APC anti-CD115 (AFS98), PE anti-Ly6C (HK1,4), PerCP anti-CD11b (M1/70), AlexaFluor488 anti-CD146 (ME-9F1), PECy7 anti-Gr1 (RB6-8C5), APC Cy7 anti-CD45 (30-F11, Biolegend). All samples were acquired with a Flow Cytometer Cyan (Beckman Coulter), and analyzed with FloJo (Treestar).
Lacotte S., Slits F., Orci L.A., Meyer J., Oldani G., Delaune V., Gonelle-Gispert C., Morel P, & Toso C. (2016). Impact of myeloid-derived suppressor cell on Kupffer cells from mouse livers with hepatocellular carcinoma. Oncoimmunology, 5(11), e1234565.
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2

Nanoparticle-mediated Cancer Immunotherapy Protocol

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DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) (890890) and cholesterol (700000) lipids were purchased from Avanti Polar Lipids, Inc., Alabaster, AL, USA. Nanoplasmid (NTC 9385R-MCS) containing the CRT gene was purchased from Aldevron (Lincoln, NE, USA). Chloroform (C2432) was purchased from Sigma-Aldrich, St. Louis, MO, USA. HEPES, sterile 1 M buffer (J848), DMEM (11965092), Fetal bovine serum, FBS (10082147), Penicillin-Streptomycin - PenStrep (15140122), PBS (10010023), Collagenase IV (17104019), were purchased from ThermoFisher/Gibco, Waltham, MA, USA. The following Fluorochrome-conjugated monoclonal antibodies (mAbs) for flow cytometry were purchased from BioLegend, San Diego, CA, USA and were listed: APC-Cy7 anti-CD45 (103115), PerCP anti-CD3 epsilon (100325), PE-Cy5 anti-CD4 (100410), PE-Cy7 anti-CD8a (100721), PE anti-Granzyme B (372208), BV510 anti-INFg (505841), BV510 anti-Ly-6G (127633), PE anti-Ly-6C (128007), BV650 anti-CD11b (101239), APC anti-CD206 (141707), BV421 anti-CD11c (117329). AF488 anti-iNOS (53-5920-82) was procured from eBiosciences/ThermoFisher, Waltham, MA, USA. Liberase (5401119001) was procured from Life Technologies, NY, USA. Transcription factor buffer set (562574) was purchased from BD Biosciences, San Jose, CA, USA. B16F10 was provided by Dr. Mary Jo Turk, Dartmouth. Aged C57BL/6 mice were obtained from NIA, Bethesda, MD, USA.
Singh A., Ashar H., Butcher J.T, & Ranjan A. (2023). Age-associated changes in the gut microbiome impact efficacy of tumor immunomodulatory treatments. Experimental gerontology, 181, 112268.
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3

Flow Cytometric Phenotyping of Myeloid Cells

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Cells were stained for flow cytometry according to standard procedures (20 (link)). Neutrophils were stained with anti-CD11b-Alexa488 and anti-Ly6C-PE or anti-Ly6G-PE (Biolegend). Macrophages were stained with anti-F4/80-Alexa647 (Biolegend). Fluorescently labeled antibodies were generally used at 1 μg/mL.
McDonald G., Cabal N., Vannier A., Umiker B., Yin R.H., Orjalo AV J.r., Johansson H.E., Han J.H, & Imanishi-Kari T. (2015). Female Bias in Systemic Lupus Erythematosus is Associated with the Differential Expression of X-Linked Toll-Like Receptor 8. Frontiers in Immunology, 6, 457.
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4

Characterizing Myeloid Cells in Trem1 Mice

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Freshly harvested tumors from Trem1+/+ or Trem1–/– mice were processed into a single-cell suspension using gentleMACS Octo Dissociator with Heaters (Miltenyi Biotech) in combination with the tumor dissociation kit (Miltenyi Biotech). Cells were stained with fluorochrome–conjugated antibodies according to the manufacturer’s instructions. For surface staining, cells were prepared and suspended in PBS and incubated with following antibodies (all from BioLegend) at 4°C for 45 minutes in dark: TruStain FcX (clone: 93, 101319, 1:50 dilution), anti-F4/80-APC (clone: BM8, 123116, 1:100 dilution), anti-F4/80-FITC (clone: BM8, 123108, 1:100 dilution), anti-CD11b-APC (clone: M1/70, 101212, 1:100 dilution), anti-CD11b-PE (clone: M1/70, 101208, 1:200 dilution), anti-CD11b-APC/Cy7 (clone: M1/70, 101226, 1:100 dilution), anti-Gr1-APC/Cy7 (clone: RB6-8C5, 108423, 1:100 dilution), anti-Ly6C-PE (clone HK1.4, 128007, 1:200 dilution), anti-Ly6C-APC/Cy7 (clone: HK1.4, 128026, 1:100 dilution), anti-Ly6G-PE (clone: 1A8, 127608, 1:200 dilution), and anti-Ly6G-APC/Cy7 (clone: 1A8, 127624, 1:100 dilution). Cells were acquired on the Attune NxT Acoustic Focusing flow cytometry platform (Thermo Fisher Scientific) and data were analyzed on FlowJo v10.0.
Ajith A., Mamouni K., Horuzsko D.D., Musa A., Dzutsev A.K., Fang J.R., Chadli A., Zhu X., Lebedyeva I., Trinchieri G, & Horuzsko A. (2023). Targeting TREM1 augments antitumor T cell immunity by inhibiting myeloid-derived suppressor cells and restraining anti–PD-1 resistance. The Journal of Clinical Investigation, 133(21), e167951.
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5

Identification of Tumor-Infiltrating Immune Cells

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Tumor cells isolated from mice were thawed and stained for identification of myeloid or lymphoid cell subpopulations according to the procedure described by Rossowska et al. (30 (link)). Briefly, tumor-derived cells were stained with LIVE/DEAD Fixable Violet Dead Staining Kit (Thermo Fisher) and then stained with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, anti-CD19 PE-CF594, anti-CD49b PE-CF594 (all from BD Biosciences), anti-CD45 BV605, anti-CD11b PerCP-Cy5.5, anti-CD11c BV650, anti-F4/80 AlexaFluor 700, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHC II FITC, anti-CD86 PE-Cy7 (all from BioLegend) for myeloid cell identification, and anti-CD45 BV605, anti-CD3 BV650, anti-CD4 FITC, anti-CD8 APC/Fire 750, anti-CD25 PE, anti-CD44 PE-Cy7, anti-CD62L PerCP-Cy5.5 (all from BioLegend) for lymphocytes identification. Then, the cells were fixed using the FoxP3 Fixation Permeabilization Staining Kit (eBioscience). Tumor cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or anti-FoxP3 APC (eBioscience) antibodies, respectively. The analysis was performed using a FACS Fortessa flow cytometer with Diva software (Becton Dickinson).
Rossowska J., Anger N., Wegierek K., Szczygieł A., Mierzejewska J., Milczarek M., Szermer-Olearnik B, & Pajtasz-Piasecka E. (2019). Antitumor Potential of Extracellular Vesicles Released by Genetically Modified Murine Colon Carcinoma Cells With Overexpression of Interleukin-12 and shRNA for TGF-β1. Frontiers in Immunology, 10, 211.
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6

Immune Cell Activation Assay

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Ultrapure LPS, flagellin, nigericin, CpG oligonucleotide, and poly(dA:dT) were purchased from InvivoGen. Silica (MIN-U-SIL 15) was obtained from US Silica. ATP was from Sigma-Aldrich. Pam3Cys was from EMC Microcollections. X-tremeGENE HP DNA transfection reagent was from Roche. Transfection reagent Profect P1 was from Targeting Systems. Acridine Orange (L13159) was from Alfa Aesar. TRIzol reagent (15596018), BAPTA-AM (B1205), and Alexa Fluor 594-labeled zymosan particles (Z23374) were from Thermo Fisher Scientific. Antibodies for immunoblotting include anti-CHMP4B (13683-1-AP) from Proteintech; anti-IKKα (05-536) from Millipore; anti-Mcu (14997), anti-phospho-IkBα (S32), anti-phospho-IKKα/β (S176/180), anti-phospho-p65(S536), anti-p65, anti-phospho-ERK1/2 (T202/Y204), anti-phospho-JNK (T183/Y185), and anti-phospho-p38 (T180/Y182) from Cell Signaling Technology; anti-β-actin (sc-1615) from Santa Cruz Biotechnology; anti-NLRP3 (Cryo-2), anti-Asc (AL177), and anti-caspase-1 (AG-20B-0042) from Adipogen; and anti-IL-1β (AF-401-NA) from R&D Systems. Antibodies for the flow cytometry assay include anti-CD45-PE Cy7 (103114), anti-CD11b-FITC (101206), anti-Ly-6G-PB (127612), and anti-Ly-6C-PE (128008) from BioLegend.
Dong H., Zhao B., Chen J., Liu Z., Li X., Li L, & Wen H. (2022). Mitochondrial calcium uniporter promotes phagocytosis-dependent activation of the NLRP3 inflammasome. Proceedings of the National Academy of Sciences of the United States of America, 119(26), e2123247119.
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7

Comprehensive Immune Profiling of Tumor and Spleen Cells

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Tumor cells and spleen cells isolated from mice were thawed and stained for identification of myeloid or lymphoid cell subpopulations according to the procedure described previously (46 (link)). Briefly, tumor single-cell suspensions were stained with the LIVE/DEAD Fixable Violet Dead Staining Kit (Thermo Fisher Scientific, Inc.) and then labelled with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, anti-CD19 PE-CF594, anti-CD49b PE-CF594 (all from BD Biosciences), anti-CD45 BV605, anti-CD11b PerCP-Cy5.5, anti-CD11c BV650, anti-F4/80 Alexa Fluor 700, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHC II FITC, anti-CD80 PE-Cy7 (all from BioLegend) for myeloid cell identification, and anti-CD45 BV605, anti-CD3 BV650, anti-CD4 FITC, anti-CD8 APC/Fire 750, anti-CD25 PE (all from BioLegend) for lymphocyte identification. Then, the cells were fixed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or anti-FoxP3 APC (eBioscience) antibodies, respectively. In spleen single cell suspension only the lymphocyte identification was performed according to the procedure described above. The analysis was performed using a FACS Fortessa flow cytometer with Diva software (Becton Dickinson).
Szczygieł A., Anger-Góra N., Węgierek-Ciura K., Mierzejewska J., Rossowska J., Goszczyński T.M., Świtalska M, & Pajtasz-Piasecka E. (2021). Immunomodulatory potential of anticancer therapy composed of methotrexate nanoconjugate and dendritic cell-based vaccines in murine colon carcinoma. Oncology Reports, 45(3), 945-962.
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8

Tumor Tissue Dissociation Protocol

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Weighed tumors were minced and allowed to digest in a mixture of 1mg/ml of collagenase P (Roche 11213857001), 0.1 mg/ml Collagenase I (Sigma, C0130), 0.1 mg/ml Dispase II (Roche, 04942078001) and DNase (5MU/ml, Calbiochem, 260913) in DMEM media at 37°C for 30 min. The tissue lysate was filtered through a 40 µm mesh prior to immunostaining. The resulting single-cell suspension was stained with fixable viability dye eFluor 780, anti-CD45-APC-Cy7 (103115, 1:200), anti-CD11b-FITC (101205, 1:100) and anti-Ly6/C-PE (128007, 1:100) (all from BioLegend). The percentage of positive cells were analyzed by FlowJo and gated on CD45 positivity. Unstained, live/dead only, and single stain served as control. Doublets were gated out using forward-scatter width/height and side-scatter width/height event characteristics.
Laklai H., Miroshnikova Y.A., Pickup M.W., Collisson E.A., Kim G.E., Barrett A.S., Hill R.C., Lakins J.N., Schlaepfer D.D., Mouw J.K., LeBleu V.S., Roy N., Novitskiy S.V., Johansen J.S., Poli V., Kalluri R., Iacobuzio-Donahue C.A., Wood L.D., Hebrok M., Hansen K., Moses H.L, & Weaver V.M. (2016). Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular-fibrosis and tumor progression. Nature medicine, 22(5), 497-505.
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9

Tumor Tissue Dissociation Protocol

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Weighed tumors were minced and allowed to digest in a mixture of 1mg/ml of collagenase P (Roche 11213857001), 0.1 mg/ml Collagenase I (Sigma, C0130), 0.1 mg/ml Dispase II (Roche, 04942078001) and DNase (5MU/ml, Calbiochem, 260913) in DMEM media at 37°C for 30 min. The tissue lysate was filtered through a 40 µm mesh prior to immunostaining. The resulting single-cell suspension was stained with fixable viability dye eFluor 780, anti-CD45-APC-Cy7 (103115, 1:200), anti-CD11b-FITC (101205, 1:100) and anti-Ly6/C-PE (128007, 1:100) (all from BioLegend). The percentage of positive cells were analyzed by FlowJo and gated on CD45 positivity. Unstained, live/dead only, and single stain served as control. Doublets were gated out using forward-scatter width/height and side-scatter width/height event characteristics.
Laklai H., Miroshnikova Y.A., Pickup M.W., Collisson E.A., Kim G.E., Barrett A.S., Hill R.C., Lakins J.N., Schlaepfer D.D., Mouw J.K., LeBleu V.S., Roy N., Novitskiy S.V., Johansen J.S., Poli V., Kalluri R., Iacobuzio-Donahue C.A., Wood L.D., Hebrok M., Hansen K., Moses H.L, & Weaver V.M. (2016). Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular-fibrosis and tumor progression. Nature medicine, 22(5), 497-505.
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10

Tumor Immune Cell Profiling

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Tumors were harvested, weighed, minced, and digested for 45 minutes with gentle shaking at 37°C, in HBSS containing 7 mg/ml Collagenase D and 2 mg/ml DNase-I (Roche). Single cell suspensions were stained with antibodies including anti-CD45-APC-Cy7, anti-CD3-Vioblue, anti-CD8-PCP, anti-CD4-PCP, anti-Foxp3-FITC, anti-CD11b-APC, CD11b-PCP, anti-Gr-1-PE-Cy7, anti-Ly6C-PE, and anti-Ly6G-PE-Cy7 (BioLegend or eBioscience); anti-CCR2-APC or control Rat IgG2B-APC antibody were obtained from R&D. Cells were fixed/permeabilized using reagents from the Foxp3 staining kit (eBioscience). Flow cytometry was performed on a Miltenyi MACSQuant 10 Analyzer. Representative gating strategies are presented in Supplementary Fig. S1. To determine absolute cell number per gram of tumor, total numbers of cells in appropriate gates were multiplied by a correction factor for the proportion of the tumor sample run through the cytometer, and normalized for total tumor weight.
Steinberg S.M., Shabaneh T.B., Zhang P., Martyanov V., Li Z., Malik B.T., Wood T.A., Boni A., Molodtsov A., Angeles C.V., Curiel T.J., Whitfield M.L, & Turk M.J. (2017). Myeloid cells that impair immunotherapy are restored in melanomas which acquire resistance to BRAF inhibitors. Cancer research, 77(7), 1599-1610.
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