Fluorescently labeled antibodies

Manufactured by BioLegend

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Fluorescently labeled antibodies are laboratory reagents used in various immunoassays and cell analysis techniques. These antibodies are conjugated with fluorescent dyes, allowing for the detection and visualization of target molecules or cells.

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Lab products found in correlation

Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions) Ctla4 ig, by BioXCell (1 mentions) Monensin, by Merck Group (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Cell culture media, by Corning (1 mentions) Anti cd3 okt3, by Thermo Fisher Scientific (1 mentions) Kod polymerase master mix, by Merck Group (1 mentions) Anti cd28 cd28.2 activating antibodies, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Corning (1 mentions) Polybrene, by Merck Group (1 mentions) Concanavalin a (cona), by Merck Group (1 mentions) Retronectin, by Takara Bio (1 mentions) Human ab serum, by Omega Scientific (1 mentions) Anti cd3 anti cd28 beads, by Thermo Fisher Scientific (1 mentions) Negative magnetic bead selection, by STEMCELL (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Cd4 pe, by BioLegend (1 mentions) Cd40 fitc, by BioLegend (1 mentions) Cd206 pe, by BioLegend (1 mentions) Cd14 fitc, by BioLegend (1 mentions)

Spelling variants of this product

Fluorescently labeled monoclonal antibodies (5 citations)

9 protocols using fluorescently labeled antibodies

Generation of CTLA-4-CD28 Fusion Protein

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Full length human CD28 cDNA (originally provided by C. Thompson MD, Memorial Sloan-Kettering Cancer Center, New York, New York) was cloned into the GFP-RV vector (provided by K. Murphy, Washington University School of Medicine, St Louis, MO) and single point mutations made using the Q5 Site Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA) A CTLA-4-CD28 fusion protein was constructed consisting of the extracellular domain of murine CTLA-4 (amino acids 1-162) and the transmembrane and cytoplasmic domain of human CD28 (amino acids 152-219). All constructs were verified by direct sequencing. Chinese Hamster Ovary cells (CHO) expressing IA^d (gift from Dr K. Murphy, Washington University School of Medicine, St Louis, MO) were stably transfected with plasmids expressing either human CD80 or CD86 (Sino Biologicals, China). PlatinumE (PlatE) packaging cell line and the retroviral packaging vector pCMV-10A1 were kindly provided by Dr. T. Egawa (Washington University School of Medicine, St Louis, MO).
Fluorescently labeled antibodies were purchased from Biolegend (San Diego, CA) unless otherwise stated. CTLA4Ig was purchased from BioXcell (West Lebanon, NH) and human CD80Ig and CD86Ig were purchased from ACRObiosystems (Newark, DE).

Gmyrek G.B., Pingel J., Choi J, & Green J.M. (2017). Functional analysis of acquired CD28 mutations identified in cutaneous T cell lymphoma. Cellular immunology, 319, 28-34.

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Flow cytometry analysis of Treg cells

Cited 2 times

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Single cell suspensions from peripheral blood, renal lymph nodes (LN) and spleen were prepared as before and analyzed for cell surface markers and intracellular Foxp3 expression by flow cytometry using fluorescently labeled antibodies (BioLegend and eBiosciences) (14 (link),20 (link)). For intracellular cytokine analysis, single cell suspensions were stimulated ex vivo for 5 hours with phorbol 12-myristate 13-acetate (PMA, 20ng/ml) and ionomycin (1μg/ml) in the presence of 1μM monensin (all from Sigma, USA). Gated CD4⁺ T cells were analyzed with a 5-color upgraded (Cytek Development) FACScan™ (BD Biosciences) equipped with CellQuest™ and Rainbow™ software for data acquisition. The data was analyzed using FlowJo™ software (FlowJo LLC).

Stremska M.E., Dai C., Venkatadri R., Wang H., Sabapathy V., Kumar G., Jose S., Mohammad S., Sung S.S., Fu S.M, & Sharma R. (2019). IL233, an IL-2-IL-33 Hybrid Cytokine Induces Prolonged Remission of Mouse Lupus Nephritis by Targeting Treg cells as a Single Therapeutic Agent. Journal of autoimmunity, 102, 133-141.

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T Cell Activation and Expansion Protocol

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Primers were purchased from Integrated DNA Technologies (Coralville, IA). KOD polymerase master mix and polybrene were purchased from EMD Millipore (Darmstadt, Germany). Sequencing was performed by Retrogen Inc (San Diego, CA). Fluorescently labeled antibodies and 7-AAD for flow cytometry were purchased from Biolegend (San Diego, CA), eBioscience (San Diego, CA), or Beckman Coulter (Brea, CA). Peptides were purchased from Anaspec Inc (Fremont, CA) or Thermo Fisher Scientific (Waltham, MA). Anti-CD3 (OKT3) and anti-CD28 (CD28.2) activating antibodies were purchased from eBioscience (San Diego, CA). Short-dated Proleukin was provided by Prometheus Laboratories Inc (San Diego, CA) through an investigator-initiated trial program. All other cytokines were purchased from Peprotech, Inc. (Rocky Hill, NJ). Concanavalin A was purchased from Sigma-Aldrich (St. Louis, MO). RetroNectin was purchased from Clontech Laboratories (Mountain View, CA). BioT transfection reagent was purchased from Bioland Scientific (Paramount, CA). Cell culture media, antibiotics, and fetal bovine serum were purchased from Corning (Corning, NY). Human AB serum was purchased from Omega Scientific (Tarzana, CA). Peptides were purchased from Anaspec (San Jose, CA).

Bethune M.T., Gee M.H., Bunse M., Lee M.S., Gschweng E.H., Pagadala M.S., Zhou J., Cheng D., Heath J.R., Kohn D.B., Kuhns M.S., Uckert W, & Baltimore D. (2016). Domain-swapped T cell receptors improve the safety of TCR gene therapy. eLife, 5, e19095.

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Modulation of CD8+ T-cell Cytokine Secretion by SERCA Inhibitors

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Example 2

CD8+ T-cells were isolated from human PBMCs using negative magnetic bead selection (Stemcell Technologies). Cells stimulated with anti-CD3/anti-CD28 beads (Life Technologies) and were treated with different SERCA inhibitors: thapsigargin (1.5 μM), cyclopiazonic acid (3 μM), and NS-1619 (30-100 μM). All incubations were done overnight. Subsequent activation was measured by cytokine release using intracellular staining flow cytometry of IFNγ and TNFα, using fluorescently labeled antibodies (Biolegend).

Secretion of the inflammatory cytokine, IFNγ, was increased by CD8+ T cells with the addition of each of the different SERCA inhibitors, as shown in Table 17 below.

TABLE 17

SECRETION OF IFNγ FROM IMMUNE CELLS TREATED WITH

SERCA PUMP INHIBITORS

Fold Change % IFNγ+ Cells

(Normalized to Bead

SampleStimulated Only)

CD8+ T-Cell Bead Stimulated Only1

CD8+ T-Cell Bead Stimulated + 2.08

Thapsigargin 1.5 μM

CD8+ T-Cell Bead Stimulated + 2.38

Cyclopiazonic Acid 3 μM

CD8+ T-Cell Bead Stimulated + 1.1

NS-1619 30 μM

CD8+ T-Cell Bead Stimulated + 2.68

NS-1619 100 μM

US11013717B1. Methods and compositions for treating cancer using SERCA pump inhibitors (2021-05-25). Flagship Pioneering Innovations V, Inc. [US]. Inventors: Avak Kahvejian [US], Jordi Mata-Fink [US], Jonathan Barry Hurov [US], Chengyi Jenny Shu [US], Eric Franklin Zhu [US], Katherine Mary Molloy [US].

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Multicolor Flow Cytometry of Immune Cell Subsets

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Cell surface staining was performed by incubating the investigated cellular fractions with fluorochrome-conjugated antibodies in the dark on ice for 30 minutes before washing with cold phosphate-buffered saline (PBS) and fixing with 10% Cell Fix (250 µL) (BD Biosciences, USA). A cocktail of fluorescently labeled antibodies (BioLegend, San Diego, CA, USA) containing anti-human CD3-FITC (clone HIT3a) and CD4-PE (clone RPA-T4); CD14-FITC (clone: 63D3), CD16-PE (clone: B73.1), and HLA-DR-APC (clone L243); or CD40-FITC (clone 5C3), CD206-PE (clone 15-2) and CD14-APC (clone M5E2) was used for staining. A minimum of 10,000 events were acquired on gated lymphocytes and monocytes on a BD FACSCalibur flow cytometer (BD Biosciences, USA), and data were analyzed using FlowJo software following the gating strategy presented in Figure 2.

Osei-Wusu S., Tetteh J.K., Musah A.B., Ntiamoah D.O., Arthur N., Adjei A., Arbues A., Ofori E.A., Mensah K.A., Galevo S.E., Frempong A.F., Asare P., Asante-Poku A., Otchere I.D., Kusi K.A., Lenz T.L., Gagneux S., Portevin D, & Yeboah-Manu D. (2023). Macrophage susceptibility to infection by Ghanaian Mycobacterium tuberculosis complex lineages 4 and 5 varies with self-reported ethnicity. Frontiers in Cellular and Infection Microbiology, 13, 1163993.

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Profiling Tumor-Infiltrating CD8+ T Cells

Cited 1 time

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Tumor samples were disposed with Tumor Dissociation Kit (Miltenyi Biotec). Then single-cell suspensions from tumors were lysed to remove red blood cells using RBC Lysis/Fixtion Solution (BioLegend) and filtered through a 70 μm MACS SmartStrainer (Miltenyi Biotec). After that, the single-cell suspensions were incubated with fluorescently labeled antibodies (BioLegend) against CD45, CD3, CD4, CD8 and PD-1 at 4℃ for 45 min. For intracellular staining, cells were fixed and permeabilized, and then incubated with TIM-3 (BioLegend), TOX (eBioscience), TCF-1 (BD) at 4℃ for 6 h. Multicolor flow cytometry analysis was conducted with BD Fortessa flow cytometer. Expression level of PD-1 on CD8⁺ T cells was gated by its low controls of CD8⁺ T cells from spleen of untreated mice (PD-1^low), high controls of cells stained by both anti-TOX and anti-PD-1(PD-1^hi) and between them (PD-1^int) [15 (link)] (Details are provided in the Supplementary Methods section). The proportion of early Tex (CD8⁺PD-1^int) and terminal Tex (CD8⁺PD-1^hi) in CD8⁺ T cells, the ratio of early to terminal Tex (E/T), and mean fluorescence intensity (MFI) of PD-1 expression on CD8⁺ T cells were further analyzed using FlowJo software (Treestar).

Zhang Y., Hu H.H., Zhou S.H., Xia W.Y., Zhang Y., Zhang J.P., Fu X.L, & Yu W. (2023). PET-based radiomics visualizes tumor-infiltrating CD8 T cell exhaustion to optimize radiotherapy/immunotherapy combination in mouse models of lung cancer. Biomarker Research, 11, 10.

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Monocyte Purity Determination

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Cell purity was determined using a flow cytometer (FACSCalibur, BD Biosciences) with fluorescently labeled antibodies (BioLegend, San Diego, CA) against CD14 (monocytes) and CD41 (platelets) with isotype controls. Preparations contained 98–100% monocytes of all mononuclear cells and the contamination of platelets varied from 10 to 27 platelets/monocyte (Fig. 3). The cells were utilized within 1–6 hours and the viability was determined to be 91–100% with propidium iodide and trypan blue exclusion assays. Cell counts in blood samples and in monocyte preparations were performed either in a hemocytometer (at 400x) or with an ABX Pentra 60 analyzer (Horiba, Japan).

Peshkova A.D., Le Minh G., Tutwiler V., Andrianova I.A., Weisel J.W, & Litvinov R.I. (2017). Activated Monocytes Enhance Platelet-Driven Contraction of Blood Clots via Tissue Factor Expression. Scientific Reports, 7, 5149.

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Characterization of hADSCs by Flow Cytometry

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Third-generation hADSCs were collected and prepared into cell suspensions after cells reached 80%-90% confluence. Cells were divided into 6 flow tubes with the number of cells adjusted to 1 × 10⁶ per tube, and the tubes were divided into the control group, CD34-PE group, CD45-FITC group, CD73-PE group, CD90-PE group, and CD105-PE group. Fluorescently labeled antibodies (Biolegend, USA) were added to each group except the control group. The samples were incubated at room temperature in the dark, washed with PBS (Gibco, USA), resuspended in 500 μl of PBS, and then detected by flow cytometry.

Chen J., Zhang Y., Liu M., Zhou Z., Li Q., Huang T., Yue Y, & Tian Y. (2022). Effect and Related Mechanism of Platelet-Rich Plasma on the Osteogenic Differentiation of Human Adipose-Derived Stem Cells. BioMed Research International, 2022, 1256002.

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Comprehensive Immune Cell Profiling of Tumor Microenvironment

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Mice were euthanized and tissues (tumor, spleen, peritoneal sacral and lumbar lymph nodes, peritoneal fluid, and blood) were collected. Tumors were digested using MACS Mouse Tumor Dissociation Kit and the gentle MACS Octo Dissociator (Miltenyi Biotec). Single cell suspensions from all organs were obtained using a 70 mm strainer. Red blood cells were lysed using RBC lysis buffer. Cell suspensions were stained with fluorescently labeled antibodies (BioLegend and BD Biosciences, see supplementary table of reagents), adding Brilliant Stain Buffer. For intracellular staining, cells were permeabilized and fixed using the Foxp3 fixation and permeabilization kit following manufacturer's instructions. For tetramer staining, samples were minimally processed prior to incubation with H-2Ld MuLV gp70 Tetramer-SPSYVYHQF antibody (MBL) and membrane antibodies. Dead cells were excluded using FVS780-fixable viability dye (BD Biosciences). Samples were analyzed using the Cytoflex LX flow cytometer (Beckman Coulter) and FlowJo was used to analyze the data.

Gonzalez-Junca A., Liu F.D., Nagaraja A.S., Mullenix A., Lee C.T., Gordley R.M., Frimannsson D.O., Maller O., Garrison B.S., Iyer D., Benabbas A., Truong T.A., Quach A., Tian M., Martinez R., Savur R., Perry-McNamara A., Nguyen D., Almudhfar N., Blanco C., Huynh C., Nand A., Lay Y.E., Magal A., Mangalampalli S., Lee P.J., Lu T.K, & Lee G. (2021). SENTI-101, a Preparation of Mesenchymal Stromal Cells Engineered to Express IL12 and IL21, Induces Localized and Durable Antitumor Immunity in Preclinical Models of Peritoneal Solid Tumors. Molecular cancer therapeutics, 20(9).

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