True stain monocyte blocker

Manufactured by BioLegend

Sourced in United States

The True-Stain Monocyte Blocker is a lab equipment product designed to block non-specific binding of antibodies to monocytes during flow cytometry analysis. It helps to reduce background staining and improve the specificity of cell surface marker detection.

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Lab products found in correlation

Brilliant stain buffer plus, by BD (6 mentions) Zombie green fixable viability kit, by BioLegend (4 mentions) Aurora spectral cytometer, by Cytek (4 mentions) Lsrfortessa flow cytometer, by BD (4 mentions) Human truestain fcx, by BioLegend (3 mentions) Trustain fcx plus, by BioLegend (3 mentions) Gentlemacs dissociator, by Miltenyi Biotec (3 mentions) Cytofix, by BD (3 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions) Brilliant stain buffer, by BD (3 mentions) Human trustain fcx, by BioLegend (3 mentions) Aurora spectral flow cytometer, by Cytek (3 mentions) Spectroflo v3, by Cytek (3 mentions) Buv395 anti cd11b m1 70, by BD (2 mentions) Bv421 anti cd11c n418, by BioLegend (2 mentions) Anti mouse cd16 cd32 clone 93, by BioLegend (2 mentions) Facsdiva software, by BD (2 mentions) Live dead blue, by Thermo Fisher Scientific (2 mentions) Perm wash buffer, by BD (2 mentions) Live dead fixable aqua, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Monocyte Blocker (3 citations) Human True-Stain Monocyte Blocker™ (2 citations)

41 protocols using true stain monocyte blocker

Flow Cytometric Analysis of BMDC Uptake

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BM-APCs were cultured with PLPs using the same protocol above, except PLGA NP and MP were labeled with customized IR700-CpG ODN 1826 (IDT) to detect cell uptake of PLPs with flow cytometry. Cells were harvested at 24 hours and stained for live/dead discrimination with Zombie Green Fixable Viability Kit (BioLegend, San Diego, CA) and blocked with anti-mouse CD16/CD32 (clone 93) and True-Stain Monocyte Blocker (BioLegend, San Diego, CA). For surface staining, BMDCs were incubated with BUV395 anti-CD11b (M1/70, BD), BV421 anti-CD11c (N418, BioLegend, San Diego, CA), PE–Dazzle 594 anti-Ly6C (HK1.4, BioLegend, San Diego, CA), and PE anti-(I-A/I-E) (M5/114.15.2, Invitrogen).

Pradhan P., Toy R., Jhita N., Atalis A., Pandey B., Beach A., Blanchard E.L., Moore S.G., Gaul D.A., Santangelo P.J., Shayakhmetov D.M, & Roy K. (2021). TRAF6-IRF5 kinetics, TRIF, and biophysical factors drive synergistic innate responses to particle-mediated MPLA-CpG co-presentation. Science Advances, 7(3), eabd4235.

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Monocyte Subset Analysis by Flow Cytometry

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PBMCs were stained for viability (Zombie Yellow, BioLegend, San Diego, CA) [23 (link)]. Anti-Fc receptor and True-stain Monocyte Blocker (BioLegend) were used to reduce nonspecific antibody and fluorophore binding before cells were labeled with antibodies. Non-monocytic cells were excluded with a “dump” channel containing CD3, CD19, CD20, CD56, and CD66b. Staining with CD14 and CD16 identified the two major human monocytic subsets. The antibodies used for IL-4 and chemokine receptor identification are listed in Table 2. Stained cells were acquired with a CytoFLEX flow cytometer (Beckman-Coulter, Brea, CA), courtesy of the Anesthesiology and Critical Care Medicine Flow Cytometry Core. CytEXPERT software v.2.0 (Beckman-Coulter) was used for analysis, and isotype control antibody-labeled cells were used to set negative gates. Data are reported as the change in mean fluorescence intensity (MFI) of the different receptors (MFI target – MFI isotype).

Becerra-Díaz M., Lerner A.D., Yu D.H., Thiboutot J.P., Liu M.C., Yarmus L.B., Bose S, & Heller N.M. (2020). Sex differences in M2 polarization, chemokine and IL-4 receptors in monocytes and macrophages from asthmatics. Cellular immunology, 360, 104252.

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Monocyte Subset Identification Protocol

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For classification viable monocyte singlets were classified into their respective subsets based upon expression of HLA-DR, CCR2, CD14, and CD16 (See Fig 1). 50 μL of blood was treated first with Human True Stain FcX and True Stain Monocyte Blocker (Biolegend) for 10 minutes, labeled with fluorescent antibodies for 30 min, followed by red blood cell removal with lysis buffer (Biolegend). Data were acquired on an Attune NxT flow cytometer within 2 hrs of blood collection.

Hernandez A.A., Foster G.A., Soderberg S.R., Fernandez A., Reynolds M.B., Orser M.K., Bailey K.A., Rogers J.H., Singh G.D., Wu H., Passerini A.G, & Simon S.I. (2020). An Allosteric Shift in CD11c Affinity Activates a Proatherogenic State in Arrested Intermediate Monocytes. The Journal of Immunology Author Choice, 205(10), 2806-2820.

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Flow Cytometric Identification of Hepatic Immune Cells

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Cells were prestained with a 1:100 dilution of Zombie Aqua (Fixable Viability Dye; BioLegend, London, UK) for 20 minutes at 4°C in the dark. After 10 minutes, an equal volume of a 1:100 dilution of TruStain FcX PLUS (anti-mouse CD16/32 antibody) and True-Stain Monocyte Blocker (BioLegend) was added. After a washing step, cells were stained with CD3e-PerCP-Cy5.5, CD19-PerCP-Cy5.5 (eBioscience, Thermo Fisher Scientific), NK1.1-PerCP-Cy5.5, CD103-PerCP-Cy5.5, F4/80-FITC, Ly6G-BV785, Ly6C-BV650 (BioLegend), SiglecF-PerCP-Cy5.5, CD45-APC-Cy7, CD11b-PE-Cy7 and Tim4-PE (BD Biosciences, Erembodegem, Belgium) for 20 minutes at 4°C in the dark. Cells were analyzed with a BD FACSAria Fusion flow cytometer (BD Biosciences) and FlowJo software (FlowJo LLC, BD Biosciences), and gated first as live CD45⁺ single cells. Subsequently, CD3e⁺, CD19⁺, NK1.1⁺, CD103⁺ and SiglecF⁺ cells were eliminated, and CD11b⁺Ly6C⁺Ly6G^- monocytes, CD11b⁺Ly6C^-F4/80⁺Tim4⁺ Kupffer cells (KCs) and CD11b⁺Ly6C^-F4/80⁺Tim4^- monocyte-derived macrophages (MoMfs) were gated.

Vanderborght B., De Muynck K., Lefere S., Geerts A., Degroote H., Verhelst X., Van Vlierberghe H, & Devisscher L. (2020). Effect of isoform-specific HIF-1α and HIF-2α antisense oligonucleotides on tumorigenesis, inflammation and fibrosis in a hepatocellular carcinoma mouse model. Oncotarget, 11(48), 4504-4520.

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Multiplexed Immune Profiling by ASAP-seq

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TSA and TSB conjugated antibodies and panels were obtained from Biolegend, see Supplementary Table 1 for a list of antibodies, clones and barcodes used for ASAP-seq. Cells were stained with barcoded (and fluorophore-conjugated where indicated) antibodies as previously described for CITE-seq^{5 (link),6 (link)}. Briefly, approximately 1.5-2 million cells per sample were resuspended in 1× CITE-seq staining buffer (2% BSA, 0.01% Tween in PBS) and incubated for 10 min with Fc receptor block (TruStain FcX, BioLegend, USA) to block FC receptor-mediated binding. Subsequently, cells were incubated with indicated antibodies or panels for 30 min at 4°C, as recommended by the manufacturer (BioLegend, USA). After staining, cells were washed 3× by resuspension in 1× CITE-seq staining buffer followed by centrifugation (300 g, 5 min at 4°C) and supernatant exchange. After the final wash, cells were resuspended in PBS and subjected to fixation and permeabilization as described in the section Cell fixation and permeabilization.
Intracellular staining was performed in fixed and permeabilized cells that were resuspended in Intracellular Staining Buffer (Biolegend, custom part number 900002577), with the addition of TruStain FcX and True Stain Monocyte blocker as recommended by the manufacturer (BioLegend).

Mimitou E.P., Lareau C.A., Chen K.Y., Zorzetto-Fernandes A.L., Hao Y., Takeshima Y., Luo W., Huang T.S., Yeung B.Z., Papalexi E., Thakore P.I., Kibayashi T., Wing J.B., Hata M., Satija R., Nazor K.L., Sakaguchi S., Ludwig L.S., Sankaran V.G., Regev A, & Smibert P. (2021). Scalable, multimodal profiling of chromatin accessibility, gene expression, and protein levels in single cells. Nature biotechnology, 39(10), 1246-1258.

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Visualization of Popliteal Lymph Nodes

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Popliteal LNs were harvested 3 days after infection with eGFP WT or ΔC15 ECTV, following isotype control or NK1.1 depletion. LNs were fixed overnight in periodate-lysine-paraformaldehyde buffer, equilibrated in 30% sucrose in PBS overnight and then embedded in Optimal Cutting Temperature medium (Sakura 4583) and frozen in liquid nitrogen cooled isopentane, as previously described.²² (link) Sixteen micron sections were cut and blocked in 0.01% Triton X-100 with 2% of FBS and donkey serum including TrueStain Monocyte Blocker (BioLegend 426101) for 1 h and then stained overnight with the following directly conjugated mAbs: GFP AF488 (clone FM2-64G, BioLegend 338007), NK1.1 AF647 (clone PK136, BioLegend 108719), Lyve-1 eFluor 450 (clone ALY7, Invitrogen 48-0443-80), ER-TR7 AF594 (Santa Cruz Biotechnology sc-73355), B220 AF700 (clone RA3-6B2, BioLegend 103232). Slides were washed in .1% Tween20 in PBS, mounted using Pro-Long Glass Antifade Mountant (Invitrogen P36984) and imaged using a Leica TCS SP8 WLL confocal microscope. Images were acquired using identical laser and HyD detector settings and scans were taken of an entire popliteal LN section using a 40x 1.30 NA objective, with a z step of 1.5 μm and a total z size of 9.0 μm; individual fields were then merged into a single image.

Peauroi E.M., Carro S.D., Pei L., Reynoso G.V., Hickman H.D, & Eisenlohr L.C. (2022). The ectromelia virus virulence factor C15 facilitates early viral spread by inhibiting NK cell contact. iScience, 25(12), 105510.

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Multicolor Flow Cytometry Immunophenotyping

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Monocytes from cell cultures were labeled with a cocktail of Alexa Fluor 488 anti-human CD14 (BioLegend San Diego, CA, USA), BUV496 anti-human HLA-DR (BD Biosciences), BV 510 anti-human CD16 (BioLegend), BV711 anti-human CD206 (BioLegend), BV605 anti-human CD33 (BioLegend), and BUV395 anti-human CD162 (BD Biosciences). True-Stain Monocyte Blocker (BioLegend) reagent was added prior to the label protocol to block the nonspecific binding of some fluorochromes to monocytes. Cells were acquired on a Cytek Aurora Spectral Cytometer (Cytek Biosciences, Fremont, CA, USA) and the data were analyzed employing OMIQ (Dotmatics, Woburn, MA, USA).

Avendaño-Ortiz J., Redondo-Calvo F.J., Lozano-Rodríguez R., Terrón-Arcos V., Bergón-Gutiérrez M., Rodríguez-Jiménez C., Rodríguez J.F., del Campo R., Gómez L.A., Bejarano-Ramírez N., Pérez-Ortiz J.M, & López-Collazo E. (2023). Thiosulfinate-Enriched Allium sativum Extract Exhibits Differential Effects between Healthy and Sepsis Patients: The Implication of HIF-1α. International Journal of Molecular Sciences, 24(7), 6234.

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Analyzing T-cell and Monocyte Markers

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Microspheres were decapsulated with 5 mM EDTA in HBSS with no Ca²⁺/Mg²⁺ at day seven after encapsulation and 2 million cells prepared for staining in RPMI with 5% foetal bovine serum. To measure the expression of PD-1 in CD4⁺ and CD8⁺ T cells and PD-L1 expression in CD14⁺CD11b⁺ cells, the following antibody panel was used: CD3-PE (clone HIT3a, Biolegend), HLA-DR-PerCP (clone L243, Biolegend), CD4-PerCP (clone OKT4, Biolegend), CD8-APC (clone SK1, Biolegend), CD11b-APC (clone ICRF44, Biolegend), CD45-APC/Cy7 (clone 2D1, Biolegend), CD14-AP/APC (clone HCD14, Biolegend), CD279-BB515 (Clone EH12.1, BD), CD-274-BB515 (Clone MIH1, BD) and True-Stain Monocyte Blocker (Biolegend, USA). Gates were defined using fluorescence minus one control after exclusion of the dead cells using Live/Dead fixable stain (ThermoFisher, UK). Gating strategy is provided in Figure 5—figure supplement 4. Cells were acquired after fixing them in 2% paraformaldehyde in HBSS for 1 hr using FACSAria (Becton Dickinson, UK) and analysed by FACSDiva software (Becton Dickinson) and Flow Jo version 10 (Treestar).

Tezera L.B., Bielecka M.K., Ogongo P., Walker N.F., Ellis M., Garay-Baquero D.J., Thomas K., Reichmann M.T., Johnston D.A., Wilkinson K.A., Ahmed M., Jogai S., Jayasinghe S.N., Wilkinson R.J., Mansour S., Thomas G.J., Ottensmeier C.H., Leslie A, & Elkington P.T. (2020). Anti-PD-1 immunotherapy leads to tuberculosis reactivation via dysregulation of TNF-α. eLife, 9, e52668.

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Comprehensive Immune Cell Profiling from PBMC, Adenoid, and Tonsil

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2 million cells per sample of PBMC and 5 million cells per adenoid or tonsil were resuspended in FACS buffer. Cells were first stained with LIVE/DEAD Blue (1:800, ThermoFisher) for 15 min at RT, washed twice and then incubated with True-Stain Monocyte Blocker (BioLegend) for 5 min. Antibodies for chemokine receptors and TCRγδ were sequentially added at RT (anti-CCR7 for 10 min, anti-CCR6, anti-CXCR5 and anti-CXCR3 together with Brilliant Stain Buffer Plus for 5 min, anti-TCRγδ for 10 min). An antibody cocktail containing the rest of the surface antibodies and Brilliant Stain Buffer Plus were then added directly to the cells and incubated for 30 min at RT in the dark (total staining volume 182uL). Cells were washed three times and stained with fluorescence conjugated streptavidin for 15 min at RT. Then, cells were washed twice and fixed in 1% paraformaldehyde for 20 min at RT before washing again and acquiring on the Aurora spectral cytometer (Cytek). Antibodies used in this assay are shown in Supplementary Table 8. The frequency of major populations was determined using FlowJo Software v10 (BD Biosciences) based on previously described manual gating strategies⁶².

Xu Q., Milanez-Almeida P., Martins A.J., Radtke A.J., Hoehn K.B., Chen J., Liu C., Tang J., Grubbs G., Stein S., Ramelli S., Kabat J., Behzadpour H., Karkanitsa M., Spathies J., Kalish H., Kardava L., Kirby M., Cheung F., Preite S., Duncker P.C., Romero N., Preciado D., Gitman L., Koroleva G., Smith G., Shaffer A., McBain I.T., Pittaluga S., Germain R.N., Apps R., Sadtler K., Moir S., Chertow D.S., Kleinstein S.H., Khurana S., Tsang J.S., Mudd P., Schwartzberg P.L, & Manthiram K. (2022). Robust, persistent adaptive immune responses to SARS-CoV-2 in the oropharyngeal lymphoid tissue of children. Research Square.

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Comprehensive Immune Cell Profiling

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Peripherial blood mononuclear cells (PBMCs) were freshly isolated from buffy coats of anonymous human healthy donors using Ficoll-Paque Premium (GE Healthcare). Single cells were isolated from fresh liver and tumour tissues by gentleMACS Dissociators (Miltenyi Biotec) following manufacturer’s instructions. Cells were collected and washed, and counted as numbers per gram of tissue. 5∗10⁶ single cells/tube were be collected and blocked with Mouse BD Fc Block (BD Biosciences) and True Stain Monocyte Blocker (BioLegend). For surface staining, the antibody mix was prepared with Brilliant Stain Buffer Plus (BD Biosciences) and incubated with the samples for 20 min at 4°C. For intracellular markers, the cells were subsequently permeablised with Transcription Factor Buffer Set (BD Biosciences) according to the manufacture’s protocol. Samples were stained with intracellular antibodies for 50 min at 4°C and washed by Perm wash buffer (BD Biosciences). Flow cytometry was performed using BD FACSAria Fusion or BD FACSymphony A5 Cell Analyzer (BD Biosciences). Spectral unmixing and high-parameter analysis was performed by FlowJo v10 Software using the UMAP, FlowSOM and ClusterExplorer plugins (BD Biosciences). The cell proportions and absolute number would be calculated. The antibodies used are listed in the key resources table.

Zhou J., Wang H., Shu T., Wang J., Yang W., Li J., Ding L., Liu M., Sun H., Wong J., Lai P.B., Tsang S.W., Ward S.E., Chow K.L., Sung J.J, & Sze-Lok Cheng A. (2023). Myeloid-intrinsic cell cycle-related kinase drives immunosuppression to promote tumorigenesis. iScience, 26(10), 107626.

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