Ly6c fitc

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Ly6C-FITC is a fluorescently labeled antibody that binds to the Ly6C antigen, which is expressed on the surface of certain immune cells. It can be used to identify and quantify these cell populations in flow cytometry or other applications.

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Anti-Ly6C-FITC (11 citations) Anti-Ly6C FITC (clone HK1.4; (3 citations) Fluorescein isothiocyanate (FITC) conjugated anti-mouse Ly6G/Ly6C (Gr-1) antibody (2 citations) FITC-conjugated anti-Ly6C (2 citations) FITC-Ly6C (HK1.4, (2 citations) FITC anti-mouse Ly6C (2 citations)

33 protocols using ly6c fitc

Multiparametric Flow Cytometry Analysis

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PE-Cy™7-CD4 (Clone: RM4-5), APC-Cy™7-CD69 (Clone: H1.2F3), APC-Cy™7-CD45 (Clone: 30-F11), APC-Cy™7-CD19 (Clone: 6D5), PE-Cy™7-MHC II (Clone: M5/114.15.2), PE-Foxp3 (Clone: MF-14), FITC-Ly6C (Clone: HK1.4), BV421-CD11b (Clone: M1/70) were purchased from Biolegend. PE-F4/80 (Clone: BM8), APC-IFN-γ (Clone: XMG1.2), Biotin-CD11c (Clone: N418) were purchased from eBioscience. APC-Cy™7-CD4 (Clone: GK1.5), APC-CD40 (Clone: HM40-3), APC-CD25 (Clone PC61), PerCP-Cy™5.5-CD80 (Clone: 16-10A1), FITC-CD3e (Clone: 145-2C11), PE-IL-17a (Clone: TC11-18H10), Sav-BUV395 were purchased from BD Biosciences. Flow cytometry data were acquired on BD LSR II flow cytometer as previously described⁴⁰ and analyzed with FlowJo™ v10.7.

Guan D., Wang Z., Huo J., Xu S, & Lam K.P. (2021). Bruton’s tyrosine kinase regulates gut immune homeostasis through attenuating Th1 response. Cell Death & Disease, 12(5), 431.

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Analyzing Peritoneal Leukocyte Subsets and Efferocytosis

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Exudate cells from the peritoneal lavages were prepared to determine the leukocyte subtypes. The cells were blocked with anti-mouse CD16/CD32 and stained with anti-mouse APC-Ly6G, e450-F4/80 (all from eBioscience, San Diego, CA, USA) and FITC-Ly6C (BioLegend, San Diego, CA, USA) for 30 min at 4 °C. To determine the efferocytosis rate in vivo, cells were permeabilized, then stained with PerCP-Cy5.5-conjugated anti-Ly6G (eBioscience) and analyzed using flow cytometry (BD FACSCanto II) (Supplementary Figure 2). Cytokines were measured in the murine peritoneal exudates using standard ELISA (R&D Systems, Minneapolis, MN, USA).

Körner A., Schlegel M., Theurer J., Frohnmeyer H., Adolph M., Heijink M., Giera M., Rosenberger P, & Mirakaj V. (2017). Resolution of inflammation and sepsis survival are improved by dietary Ω-3 fatty acids. Cell Death and Differentiation, 25(2), 421-431.

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Lipid-Based Nanoparticle Preparation and Characterization

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The lipids 18: 1TAP (1, 2-dioleoyl-3-trimethylammonium-propane), DOPE (1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine), EPC, 16:0 NBD PE, and 16:0 Liss Rhod PE were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). DSPE-PEG2000-Folic acid was purchased from Nanocs (Boston, MA, USA). Chol (Cholesterol), Collagenase, and DNase I were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The dye DiI was purchased from Beyotime Biotechnology. PTX was purchased from Meilunbio^® (Dalian, China). Paclitaxel injection was purchased from HAPHARM Group Co., Ltd. (Harbin, China). The CD9, CD63, and TSG101 antibodies were purchased from Abcam (Cambridge, MA, USA).
The antibodies of flow cytometry (anti-mouse): PE-CD45, FITC-CD4, APC-CD8a, PerCP/Cyanine5.5-CD69, FITC-CD3ε, APC-CD49b (pan-NK cells), APC/Cyanine7-CD335 (NKp46), PE-CD107a (LAMP-1), APC-CD45R/B220, PE/Cyanine7-CD69, FITC-CD45, APC/Cyanine7-F4/80, PE-CD11c, PE/Cyanine7-CD86, APC anti-mouse/human CD11b, PerCP/Cyanine5.5-Ly-6G, FITC-Ly-6C, PE/Cyanine7-CD45, APC-CD25, PE-FOXP3, PE-CD86, and PerCP/Cyanine5.5-CD206 (MMR) were purchased from Biolegend (San Diego, CA, USA).

Wang X., Li D., Li G., Chen J., Yang Y., Bian L., Zhou J., Wu Y, & Chen Y. (2024). Enhanced Therapeutic Potential of Hybrid Exosomes Loaded with Paclitaxel for Cancer Therapy. International Journal of Molecular Sciences, 25(7), 3645.

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Multiparameter Flow Cytometry Panel

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APC: anti-Ly6G (clone: 1A8, Biolegend); APCeF780: CD8 (clone: 53–6.7, eBioscience), CD45 (clone: 30-F11, eBioscience), CD24 (clone: M1/69, eBioscience); BV421: CXCR4 (clone: L276F12, Biolegend); eF450: CD11b (clone: M1/70, Invitrogen); FITC: Ly6C (clone: HK1.4, Biolegend); PE: CXCR2 (clone: SA044G4, Biolegend), SiglecF (clone: E50–2440, BD bioscience); PECy5: CD19 (clone: 1D3, eBioscience); PECy7: CD4 (clone: GK1.5, eBioscience); PEeF610: CD11c (clone N418, eBioscience); PEVio770: CD64 (clone: REA286, Miltenyi Biotec); PerCPeF710: MHCII (clone: M5/114.15.2, eBioscience).

Kulkarni U., Zemans R.L., Smith C.A., Wood S., Deng J.C, & Goldstein D.R. (2019). Excessive neutrophil levels in the lung underlie the age-associated increase in influenza mortality. Mucosal immunology, 12(2), 545-554.

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Multicolor Flow Cytometry Panel

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FITC-Granzyme B (515403), FITC-Ly6C (128006), PE-PD-L1 (124308), BV421-F4/80 (123124), APC-NK1.1 (108710), APC-CD11c (117310), APC-CD44 (103012), APC-Cy7-CD19 (115530), APC-Cy7-I-A/I-E (107628), PE-Cy7-CD8a (100722), PE-Cy7-CD206 (141720), BV605-CD4 (100548), BV605-CD11b (101237), BV711-CD69 (104537), BV711-CD45R/B220 (103255), BV711-Tbet (644819), Percp-Cy5.5-CD45 (147706) were purchased from BioLegend (San Diego, California). PE-CD62L (12-0621-82), PE-Foxp3 (12-5773-82), Pacific Blue-Ki67 (48-5698-82), APC-eFluor780-PD1 (47-9985-80), PE-Cy5-CD3 (15-0031-81), PE-Cy5-Eomes (15-4875-80), anti-CD16/32 (14-0161-85) were purchased from Thermo Fisher (Waltham, Massachusetts).

Xin B., Yang M., Wu P., Du L., Deng X., Hui E, & Feng G.S. (2022). Enhancing the Therapeutic Efficacy of PD-L1 Antibody for Metastasized Liver Cancer by Overcoming Hepatic Immunotolerance. Hepatology (Baltimore, Md.), 76(3), 630-645.

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Multi-Marker Flow Cytometry Analysis

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Cells were washed and resuspended at a density of ≤10⁶ cells/25 µl flow buffer, and Fc receptors were blocked by adding 10 µg ml^–1 TruStain FcX™ blocking solution (BioLegend). Cells were then stained in a 50 µl final volume in 96-well round-bottom plates (Corning) covered for 30 min at 4°C and washed twice with flow buffer (1× PBS, 2% FBS, 1% sodium azide) before resuspension in 150 µl of BD™ Stabilizing Fixative (BD Biosciences) and transfer to polystyrene tubes (12×75 mm) (Becton Dickinson). A total of 0.5×10⁵ to 1×10⁵ events were acquired on a BD FACSCanto™ flow cytometer with FACSDiva™ software (BD Biosciences) and analyzed by FlowJo. Fluorophore-conjugated antibodies were APC-CD45 (BD Biosciences) and BV421-CD11c, BV421-CD8, FITC-Ly6C, PE-F4/80, PE-CD31, PerCP-CD3, PerCP-CD19, PE/Cy7-Ly6G, APC/Cy7-CD11b, and APC/Cy7-CD4 (BioLegend) (antibody details in Table S6).

Figueroa-Romero C., Guo K., Murdock B.J., Paez-Colasante X., Bassis C.M., Mikhail K.A., Raue K.D., Evans M.C., Taubman G.F., McDermott A.J., O'Brien P.D., Savelieff M.G., Hur J, & Feldman E.L. (2019). Temporal evolution of the microbiome, immune system and epigenome with disease progression in ALS mice. Disease Models & Mechanisms, 13(2), dmm041947.

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Immune Cell Profiling in Blood and Spinal Cord

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Fluorophore-conjugated antibodies used as follows: BV421-CD8 (catalog 100738), FITC-CD3 (catalog 100204), FITC-Ly6C (catalog 128006), PE-NK1.1 (catalog 108708), PerCP-5.5-CD3 (catalog 100326), PerCP-5.5-CD19 (catalog 115532), APC-CD45 (catalog 103112), PE/Cy7-CD49b (catalog 108921), PE/Cy7-Ly6G (catalog 127617), APC/Cy7-CD4 (catalog 100414), and APC/Cy7-CD11b (catalog 101226) (BioLegend). Separate gating strategies identified immune cells in the peripheral blood (Supplemental Figure 4) and spinal cord (Supplemental Figure 5) as previously described (19 (link)). In the spinal cord, monocyte and microglia populations were distinguished based on CD45 levels because microglia have been shown to have low CD45 expression (59 (link)).

Murdock B.J., Famie J.P., Piecuch C.E., Pawlowski K.D., Mendelson F.E., Pieroni C.H., Iniguez S.D., Zhao L., Goutman S.A, & Feldman E.L. (2021). NK cells associate with ALS in a sex- and age-dependent manner. JCI Insight, 6(11), e147129.

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Multiparameter Flow Cytometry Panel

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Characterizing Cardiac Immune Populations

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Heart tissue was minced and enzymatically digested using collagenase I (450 U ml⁻¹), collagenase XI (60 U ml⁻¹), DNAse and hyaluronidase (60 U ml⁻¹) (Sigma-Aldrich). Single-cell suspensions were stained with CD45-PerCP/Cy5.5 (clone 30-F11, 1:600, 103132, BioLegend), CD64-APC (clone X54–5/7.1, 1:600, 139305, BioLegend), Ly6G-PE/Cy7 (clone 1A8, 1:600, 127617, BioLegend), Ly6C-FITC (clone HK1.4, 1:600, 128006, BioLegend), CD11b-BV510 (clone M1/70, 1:600, 101245, BioLegend) and DAPI (0.1%, F10347, Thermo Fisher Scientific). Blood samples from Ly6G^Tdtomato mice were stained with Ly6G-PE/Cy7 (clone 1A8, 1:600, 127617, BioLegend), CD115-BV605 (clone AFS98, 1:600, 135517, BioLegend), CD11b-APC (clone M1/70, 1:600, 101212, BioLegend), CD45-BV711 (30-F11, 1:600, 103147, BioLegend), CD3-APC/Cy7 (clone 17A2, 1:200, 100221, BioLegend), CD19-APC/Cy7 (clone 6D5, 1:300, 115529, BioLegend), B220-APC/Cy7 (clone RA3–6B2, 1:300, 103224, BioLegend), Nk1.1-APC/Cy7 (clone PK136, 1:300, 108724, BioLegend) and DAPI. Data were recorded on an LSRII flow cytometer with FACSDiva 6.1 and analyzed with FlowJo 10 software (BD Biosciences). For qRT–PCR measurements, cells were flow sorted on a FACSAria II (BD Biosciences) into 350 μl of lysis buffer (RNeasy Plus Micro Kit, Qiagen).

Grune J., Lewis A.J., Yamazoe M., Hulsmans M., Rohde D., Xiao L., Zhang S., Ott C., Calcagno D.M., Zhou Y., Timm K., Shanmuganathan M., Pulous F.E., Schloss M.J., Foy B.H., Capen D., Vinegoni C., Wojtkiewicz G.R., Iwamoto Y., Grune T., Brown D., Higgins J., Ferreira V.M., Herring N., Channon K.M., Neubauer S., Sosnovik D.E., Milan D.J., Swirski F.K., King K.R., Aguirre A.D., Ellinor P.T, & Nahrendorf M. (2022). Neutrophils incite and macrophages avert electrical storm after myocardial infarction. Nature cardiovascular research, 1(7), 649-664.

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Multiparameter Flow Cytometry of Lung Immune Cells

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Bronchoalveolar lavage was performed, and cells were removed via centrifugation. Cells were stained then fixed in IC fixation buffer (eBioscience, 00-8222-49, San Diego, CA) and run and identified via Zombie NIR (Biolegend, 423105, San Diego, CA), CD45, Alexa Fluor 532 (eBioscience, 58-0459-42, San Diego, CA), CD11c, PE-Cy7 (Biolegend, 117317, San Diego, CA), CD11b, BV480 (BD Biosciences, 566117, San Jose, CA), SIGLEC F, AF700 (eBioscience, 56-1702-80, San Diego, CA), Ly-6c, FITC (Biolegend, 128005, San Diego, CA), and Ly-6G, BV650 (Biolegend, 127641, San Diego, CA) on a Cytek Aurora Borealis at the University of Virginia flow cytometry core. Neutrophils are Zombie NIR⁻, CD45⁺, CD11c⁻, CD11b⁺, and Ly-6G⁺; eosinophils are Zombie NIR⁻, CD45⁺, CD11c⁻, CD11b⁺, and SIGLEC F⁺; inflammatory monocytes are Zombie NIR⁻, CD45⁺, CD11c⁻, CD11b⁺, Ly-6G⁻, and Ly-6C⁺⁺. Data analysis and figure generation were performed in Omiq and Graphpad Prism. A two-tailed Student’s t test was used to determined statistical significance.

Moreau G.B., Burgess S.L., Sturek J.M., Donlan A.N., Petri WA J.r, & Mann B.J. (2020). Evaluation of K18-hACE2 Mice as a Model of SARS-CoV-2 Infection. The American Journal of Tropical Medicine and Hygiene, 103(3), 1215-1219.

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