Fluorophore conjugated antibody

Manufactured by BioLegend

Sourced in United States

Fluorophore-conjugated antibodies are laboratory reagents that combine a specific antibody with a fluorescent dye, or fluorophore. This allows the antibody to be detected and visualized in various experimental techniques, such as flow cytometry, immunofluorescence, and Western blotting. The fluorophore emits light when excited by a specific wavelength, enabling the identification and quantification of the target molecule or cell population.

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Lab products found in correlation

Lsrii flow cytometer, by BD (5 mentions) Percoll, by GE Healthcare (3 mentions) Lsr 2 flow cytometer, by BD (2 mentions) Facsdiva software, by BD (2 mentions) Syrian hamster igg, by Jackson ImmunoResearch (2 mentions) 70 μm cell strainer, by BD (2 mentions) Flowlogic, by Inivai Technologies (2 mentions) Lsrfortessa, by BD (2 mentions) Macsquant10 flow cytometry machine, by Miltenyi Biotec (2 mentions) Live dead fixable blue dead cell stain kit, by Thermo Fisher Scientific (1 mentions) 40 μm strainer, by Thermo Fisher Scientific (1 mentions) Anti cd16 cd32 fc block, by BioXCell (1 mentions) Anti cd16 32 antibody clone 2.4g2, by BD (1 mentions) Collagenase 4, by Thermo Fisher Scientific (1 mentions) 7 aad viability staining solution, by BioLegend (1 mentions) Cd69 clone h1.2f3, by BioLegend (1 mentions) Dnase 1, by STEMCELL (1 mentions) Cd25 clone pc61, by BioLegend (1 mentions) Hiseq 4000, by Illumina (1 mentions) Countess 2 fl, by 10x Genomics (1 mentions)

27 protocols using fluorophore conjugated antibody

Multicolor Flow Cytometry of Kidney Cells

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Kidney cells were harvested and incubated with the following fluorophore-conjugated antibodies (BioLegend): Pacific Blue–anti-CD45, brilliant violet 650–anti-Ly6G, APC/Cy7–anti-F4/80, and APC–anti-Ly6C. Cells were blocked with anti-mouse CD16/32 antibody at 1:50 dilution for 30 min at 4°C, resuspended, and added to antibody pellets followed by fixation with 2% paraformaldehyde. Analysis was performed using an LSRII flow cytometer (BD Biosciences).

Stawowczyk M., Naseem S., Montoya V., Baker D.P., Konopka J, & Reich N.C. (2018). Pathogenic Effects of IFIT2 and Interferon-β during Fatal Systemic Candida albicans Infection. mBio, 9(2), e00365-18.

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Multiparametric Immune Checkpoint Profiling

Cited 1 time

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For PD-L1, B7-H3, and B7x expression, cells pretreated at the indicated conditions were stained with APC anti-hPD-L1 (clone 29E.2A3), PE/Cy7 anti-hB7-H3 (clone MIH42), PE anti-hB7x (clone MIH43), or isotype controls for 30 min at 4 °C. Fluorophore-conjugated antibodies were purchased from BioLegend. The staining of HHLA2 was performed by first incubating cells with primary mouse anti-hHHLA2 mAb (clone B5B5) [13 (link)] or control mouse IgG1 (clone MOPC-21) for 40 min at 4 °C, followed by staining with APC polyclonal goat F(ab′)2 anti-mouse IgG Fc (eBioscience) for 30 min at 4 °C. Cells were washed after staining and analyzed on an LSR II (BD Biosciences, San Jose, CA, USA). Data were analyzed with FlowJo (FlowJo, LLC, Ashland, OR, USA).

Wang F., Cali Daylan A.E., Deng L., Yang J., Sharma J., Su C., Li S., Zang X., Halmos B., Borczuk A, & Cheng H. (2023). Heterogeneous Expression of PD-L1, B7x, B7-H3, and HHLA2 in Pulmonary Sarcomatoid Carcinoma and the Related Regulatory Signaling Pathways. Cancers, 15(13), 3372.

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Flow Cytometry Analysis of Dendritic Cells

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Mandibular dLNs of CD11c-YFP mice were excised, and single cells were obtained by mechanically passing the samples through a 40 μm strainer (Thermo Fisher Scientific, Waltham, MA, United States). Single-cell suspensions were then blocked in FACS buffer containing 1% anti-CD16/CD32 Fc block (Bio X Cell) and were stained with a viability marker (LIVE/DEAD Fixable Blue Dead Cell Stain kit, Thermo Fisher Scientific) for 30 min at RT. Next, samples were stained with fluorophore-conjugated antibodies against CD45, CD11c, dendritic cell inhibitory receptor 2 (DCIR2), CD68, CD3, or respective isotype controls (all BioLegend, or BD Biosciences, San Jose, CA, United States) for 45 min at RT to yield fluorescence minus one stainings. After washing, samples underwent flow cytometric acquisition using a BD LSR II flow cytometer (BD Biosciences). Postacquisition data analysis was performed using FlowJo v9 software (FlowJo LLC, Ashland, OR, United States).

Lopez M.J., Seyed-Razavi Y., Yamaguchi T., Ortiz G., Sendra V.G., Harris D.L., Jamali A, & Hamrah P. (2020). Multiphoton Intravital Microscopy of Mandibular Draining Lymph Nodes: A Mouse Model to Study Corneal Immune Responses. Frontiers in Immunology, 11, 39.

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Isolation and Analysis of Immune Cells from CNS

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A single cell suspension was obtained by chopping the tissue and then forcing the dissociated CNS tissue through a 70 μm cell strainer (Falcon, United States) in HBSS supplemented with 2% fetal bovine serum (FBS). Myelin clearance was obtained by centrifugation on 37% Percoll (GE Healthcare Bio-Science AB) followed by aspiration of the myelin layer. Cells were incubated in blocking solution containing HBSS, 2% FBS, anti-CD16/32 antibody (Clone 2.4G2, BD Biosciences), Syrian hamster IgG (50 μg/ml, Jackson ImmunoResearch Laboratories Inc.) and 0.01% sodium azide, and then labeled with fluorophore-conjugated antibodies (BioLegend): anti-CD45 (clone 30-F11), CD11b (M1/70), F4/80 (BM8), GR-1 (RB6-8C5), NK1.1 (PK136), CD4 (GK1.5), and TCRβ (H57-597). Fluorescence data were acquired on an LSRII flow cytometer (BD Biosciences) with FACSDiva software (BD Biosciences) and analyzed with Flowlogic (Inivai Technologies).

Dieu R.S., Wais V., Sørensen M.Z., Marczynska J., Dubik M., Kavan S., Thomassen M., Burton M., Kruse T., Khorooshi R, & Owens T. (2021). Central Nervous System-Endogenous TLR7 and TLR9 Induce Different Immune Responses and Effects on Experimental Autoimmune Encephalomyelitis. Frontiers in Neuroscience, 15, 685645.

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Profiling Tumor-Infiltrating Lymphocytes by Flow Cytometry

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Single-cell suspensions were generated from spleens, inguinal lymph nodes, and tumors. To isolate tumor-infiltrating lymphocytes (TIL), density gradient centrifugation using an 80%/40% Percoll (GE Healthcare) gradient was done on the single-cell suspensions from excised tumors. Cells were then stained with fluorophore-conjugated antibodies from BioLegend and BD Biosciences and analyzed using an LSR II flow cytometer (BD Biosciences). Antibodies against the following cell surface markers (clone) were used: CD19 (6D5), B220 (RA3–6B2), IgD (11–26c.2a), IgM (RMM-1), IgG (Poly4053), CD80 (16–10A1), MHC II (M5/114.15.2), CD27 (LG.3A10), PD-L2 (TY25), CD1d (1B1), CD5 (53–7.3), CD138 (281–2), CD45.2 (104). Additionally, a live/dead Zombie Yellow stain and fluorophore-conjugated streptavidin with biotinylated-ovalbumin at approximately 1:10 molar ratio were used. For the apoptosis assay, a flow cytometry-based kit (BioLegend) utilizing Annexin V, propidium iodide, and select cell surface markers were used following manufacturer’s protocol. Flow cytometry data were then analyzed using FlowJo v10 (BD).

Kim S.S., Shen S., Miyauchi S., Sanders P.D., Franiak-Pietryga I., Mell L.K., Gutkind J.S., Cohen E.E., Califano J, & Sharabi A.B. (2020). B-cells improve overall survival in HPV-associated squamous cell carcinomas and are activated by radiation and PD-1 blockade.. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(13), 3345-3359.

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Isolation and Characterization of Mouse Tumor Cells

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Mouse tumors were collected, cut into small pieces (1–3mm in diameter), and digested with a mixture of Collagenase IV (100U/ml#17104019, Thermo Fisher) and DNAse I (10 μg/mL, #07469, StemCell Technologies). Tumors were incubated for 2h at 37°C, centrifuged, and filtered through a 100μm strainer. Red Blood Lysis solution was applied (#118–156-101, Quality Biological). Cells were then resuspended at a density of 1X10⁶/ml in flow cytometry binding buffer and stained with the following fluorophore-conjugated antibodies (Biolegend): CD8 (clone 53–6.7), Cxcr3 (clone CXCR3–173), Cd69 (clone H1.2F3), and Cd25 (clone PC61). 7-AAD Viability Staining Solution was used following the manufacturer’s instructions (#420403, Biolegend). Guava easyCyte 11HT Benchtop Flow Cytometer was utilized for the acquisition, and results were analyzed through FlowJo™ v10.8 Software (BD Life Sciences). The gating strategy for mouse samples flow-cytometric analysis is reported in Supplementary Fig. S2.

Romano G., Paradiso F., Li P., Shukla P., Barger L.N., Naggar O.E., Miller J.P., Liang R.J., Helms T.L., Lazar A.J., Wargo J.A., Taraballi F., Costello J.C, & Kwong L.N. (2023). Microparticle-delivered Cxcl9 prolongs Braf inhibitor efficacy in melanoma. Cancer immunology research, 11(5), 558-569.

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Single-cell RNA sequencing of tumor-infiltrating B cells

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B-cells from inguinal or tumor-draining lymph nodes were pooled from 3 different mice to obtain single-cell suspensions. Cells were then stained with fluorophore-conjugated antibodies from BioLegend and sorted on a FACSAria cell sorter. Antibodies against the following cell surface marker (clone) were used: CD19 (1D3/CD19), CD45.2 (104), CD3e (145–2C22), Gr-1 (RB6–8C5), CD11b (M1/70), TER-119 (TER-119). Cells were additionally stained with propidium iodide. Following sorting, cells were counted using an Invitrogen Countess II FL and loaded into the 10x-Genomics Chromium controller. 10x-Genomics v3 libraries were then prepared as per manufacturer recommendations. Initial quality control was done using a Qubit 2.0 Fluorometer. Libraries were then sequenced aiming for a minimum coverage of 20000 reads per cell using an Illumina HiSeq 4000 at the Institute for Genomic Medicine at the University of California, San Diego. Single-cell RNA sequencing data were initially analyzed using Cell Ranger (10x Genomics) and converted into matrix files. The data were then subsequently analyzed using Seurat v3.(49 (link),50 (link))

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Multicolor Flow Cytometry of T Cell Subsets

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APC- or PE-conjugated MHC class I tetramers were generated as described before [74 (link)]. Recombinant murine IL-15 protein was purchased from eBioscience. Fluorophore-conjugated antibodies were purchased from BioLegend (Lucerna Chem AG, Luzern, Switzerland), eBiosciences (Thermo Fisher Scientific, MA, USA) or BD Biosciences (Allschwil, Switzerland). The following antibodies (clone) were used for Flow cytometry or fluorescence microscopy: anti-CD8α (53–6.7), anti-CD8β.2 (53–6.8), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD90.1 (Ox-7), anti-CD90.2 (30-H12), anti-CD62L (MEL-14), anti-CD44 (IM7), anti-KLRG-1 (2F1), anti-Ki-67(SolA15), anti-Bcl-2 (BCL/10C4), anti-CD127 (A7R34), anti-CD4 (RM4-5), anti-CD3ε (145-2-C11), anti-CD69 (H1.2F3), anti-CD103 (2E7), anti-NKp46 (29A1.4), anti-CD49b (DX5), anti-NK1.1 (PK136), anti-CD31 (MEC13.3), anti-Podoplanin (8.1.1), anti-EpCAM (G8.8), anti-pSTAT5 (pY694). Live/Dead Fixable near-IR (Life Technologies) dead cell stain was used to exclude dead cells.

Baumann N.S., Torti N., Welten S.P., Barnstorf I., Borsa M., Pallmer K., Oduro J.D., Cicin-Sain L., Ikuta K., Ludewig B, & Oxenius A. (2018). Tissue maintenance of CMV-specific inflationary memory T cells by IL-15. PLoS Pathogens, 14(4), e1006993.

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Multiparametric Flow Cytometry Analysis

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Single cell suspensions were assessed for viability with Trypan Blue and manually counted, then incubated with Fc receptor blocking solution followed by fluorophore-conjugated antibodies (BioLegend). For intracellular staining, cells were permeabilised and stained with appropriate antibody per manufacturer’s instructions (eBioscience). Flow cytometry was performed on an LSRII (BD Biosciences) and analysed using FloJo Version X (Tree Star). See online supplementary materials for list of antibodies.

Nywening T.M., Belt B.A., Cullinan D.R., Panni R.Z., Han B.J., Sanford D.E., Jacobs R.C., Ye J., Patel A.A., Gillanders W.E., Fields R.C., DeNardo D.G., Hawkins W.G., Goedegebuure P, & Linehan D.C. (2017). Targeting both tumour-associated CXCR2+ neutrophils and CCR2+ macrophages disrupts myeloid recruitment and improves chemotherapeutic responses in pancreatic ductal adenocarcinoma. Gut, 67(6), 1112-1123.

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Isolation and Analysis of Immune Cells

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Single cells from the spleen were isolated by homogenization and red blood cell lysis using ammonium chloride solution (StemCell, USA). Mononuclear cells from the CNS were isolated as follows. Mice were deeply anesthetized under isoflurane and transcardially perfused with ice cold PBS. Brain was dissected from the skull, and spinal cord was dissected from the spinal column and placed on ice, followed by Dounce homogenization in ice cold PBS. The cell suspension was washed and loaded onto a 37%/70% Percoll gradient, followed by centrifugation. Mononuclear cells were isolated from the Percoll interphase, washed, and processed for staining.
Cells were incubated with Live-Dead fixable stain (either UV-Blue or NearIR; Life Technologies, USA), followed by surface staining using fluorophore-conjugated antibodies (Biolegend, USA) against the following markers in various combinations: CD11b, CD11c, CD45, CD45.1, CD45.2, TCRβ, CD4, CD8, CD19, CX3CR1, Ly6C, and Ly6G, followed by fixation in 1% paraformaldehyde. Cells were processed using an LSRII flow cytometer (BD Biosciences, USA). Flow cytometry analysis was performed using FlowJo software v10 (BD Biosciences, USA).

McGill M.M., Richman A.R., Boyd J.R., Sabikunnahar B., Lahue K.G., Montgomery T.L., Caldwell S., Varnum S., Frietze S, & Krementsov D.N. (2021). p38 MAP Kinase Signaling in Microglia Plays a Sex-Specific Protective Role in CNS Autoimmunity and Regulates Microglial Transcriptional States. Frontiers in Immunology, 12, 715311.

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