Trustain fcx antibody

Manufactured by BioLegend

Sourced in United States

TruStain FcX antibody is a Fc blocking reagent that can be used to block Fc receptors on cells. It helps to minimize non-specific antibody binding to Fc receptors, which is important for accurate cell surface staining and analysis.

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22 protocols using trustain fcx antibody

Multicolor Flow Cytometry Analysis

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The mouse spleens were digested with 1 mg/mL collagenase I (Gibco) in Hank’s balanced salt solution (HBSS; Life Technologies) and stained. The bone marrow (BM) was prepared from femur and BD Pharm Lyse (BD Biosciences) was added to lyse red blood cells. TruStain FcX antibody (BioLegend, San Diego, CA) was applied to block non-specific binding for 10 min at 4 °C in FACS buffer (Ca²⁺/Mg²⁺-free PBS with 1% human bovine serum albumin, 4% FBS, and 0.5 M EDTA) before staining (30 min) with appropriate antibodies. For intracellular staining, SVCs isolated from epididymal white adipose tissue (eWAT) were stimulated for 5 h at 37 °C with PMA, ionomycin, and GolgiStop (BD). Stimulated cells were washed with PBS, fixed, and permeabilized by Cytofix/Cytoperm kit (BD) as per the manufacturer’s protocol. Abs were purchased from BioLegend or R&D Systems: For mouse, CD45R/B220 (30-F11), F4/80 (BM8), Ly-6C (HK1.4), Ly-6G (1A8), CD3 (17A2), CCR7 (4B12), CD8a (53-6.7), CD11b (FAB1124S), CD4 (FAB554S), DARC (FAB6695A), CD206 (C068C2), IL-22Ra1 (FAB42941P), IL-22 (Poly5164), and IL-10 (JES5-16E3); for human, CD4 (RPA-T4), CD8 (SK1), CD14 (63D3), CD11b (ICRF44), CD16 (3G8), DARC (Clone #358307), IL-22Ra1 (Clone #305405), CD86 (IT2.2), and CD206 (15-2). Isotype control forward- and side-scatter parameters were used to remove the cell aggregates and debris.

Kim E.Y., Noh H.M., Choi B., Park J.E., Kim J.E., Jang Y., Lee H.K, & Chang E.J. (2019). Interleukin-22 Induces the Infiltration of Visceral Fat Tissue by a Discrete Subset of Duffy Antigen Receptor for Chemokine-Positive M2-Like Macrophages in Response to a High Fat Diet. Cells, 8(12), 1587.

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Tumor Immune Profiling by Flow Cytometry

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Tumors were minced and digested with 5 mL of 2 mg/mL collagenase D (Sigma-Aldrich) and 0.2 mg/mL DNase I (Sigma-Aldrich) in DMEM with 5% FBS for 1 h at 37°C to generate single cell suspensions. Cells were then filtered through a 40 μM strainer and collected by centrifugation. Cell pellets were suspended and lysed in red blood cell lysis buffer (BioLegend) for 5 min, followed by incubation with TruStain fcX antibody (BioLegend) to block nonspecific staining of antibodies. A portion of cells was used to co-stain with PE-conjugated PD-L1 antibody (BioLegend), APC-conjugated CD45 antibody (BioLegend) and 7-AAD (BioLegend). The remaining cells were fixed and permeabilized using the Fixation buffer (BioLegend) and Intracellular Staining Permeabilization Wash Buffer (BioLegend), respectively, according to the manufacturer’s protocol. The cells were then co-stained with antibodies against CD3 (BioLegend, APC conjugated), granzyme B (BioLegend, FITC-conjugated) and IFNγ (BioLegend, PE-conjugated), or co-stained with antibodies against CD3 (BioLegend, APC conjugated), CD4 (BioLegend, FITC-conjugated) and CD8 (BioLegend, PE-conjugated). The corresponding isotype IgG controls (BioLegend) were used. The cells were incubated with corresponding antibodies for 30 min in Cell Staining Buffer (BioLegend). Cells were washed with the Cell Staining Buffer and analyzed by flow cytometry.

Mathew A.G., Saxton A.M., Upchurch W.G, & Chattin S.E. (1999). Multiple Antibiotic Resistance Patterns of Escherichia coli Isolates from Swine Farms. Applied and Environmental Microbiology, 65(6), 2770-2772.

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Multiparameter Flow Cytometry Analysis

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Blood samples were collected in EDTA tubes (Sarstedt) from tail bleeds, were labeled with leucocyte-specific antibodies as previously described^{47 (link)}. Spleen was enzymatically digested for 30 min at 37 °C in Hank’s balanced salt solution (Life Technologies) containing 1 mg/ml collagenase D (Sigma) and pushed through a 70-μm cell strainer to obtain a single-cell suspension, which was then stimulated and/or stained. BM was flushed out of femur by use of a 27-gauge needle attached to a 10 ml syringe filled with PBS. Red blood cells were lysed with BD Pharm Lyse (BD Biosciences). Non-specific binding was blocked with TruStain FcX antibody (BioLegend) for 10 min at 4 °C in FACS buffer (Ca²⁺/Mg²⁺-free PBS with 2% FBS and 0.5 M EDTA) before staining (30 min) with appropriate antibodies. Abs were purchased from BioLegend and included.
CD45 (30-F11), CD11b (M1/70), Ly-6C (HK1.4), Ly-6G (1A8) CD115 (AFS98), class II major histocompatibility complex (MHC) (IA/IE), CD11c (N418), CD64 (X54-5/7.1), TCRγδ (GL3), CD3ε (145-2C11). Isotype controls Abs were used at the same protein concentrations as their corresponding markers. As a gating strategy we used forward- and side-scatter parameters to exclude cell aggregates and debris from analysis. Cells and beads (polyscience) were counted on an Attune Cytometer (Life Technologies) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Bouchareychas L., Grössinger E.M., Kang M, & Adamopoulos I.E. (2018). γδTCR regulates production of interleukin-27 by neutrophils and attenuates inflammatory arthritis. Scientific Reports, 8, 7590.

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Purification and Phenotyping of Murine Peritoneal Macrophages

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We plated peritoneal macrophages on untreated Petri dishes and allowed them to adhere overnight. The next morning, we lifted cells by incubating for 5 min in ice-cold Dulbecco's phosphate-buffered saline (DPBS; Thermo Fisher Scientific) without calcium or magnesium and scraping gently. We next filtered cells through a 40 μm cell strainer, washed them with DPBS, and resuspended them in DPBS containing 5% FBS, 10 mM HEPES, 1 mM EDTA, and TruStain fcX™ antibody (BioLegend, San Diego, CA). We stained 10⁵ to 10⁶ cells with APC anti-mouse CD36 (clone: HM36; BioLegend), PE/Cy7 anti-mouse CD64 (clone: ×54–5/7.1; BioLegend), and PE anti-mouse F4/80 (clone: BM8; eBioscience) antibodies for 20 min on ice. We then washed and resuspended cells in DPBS and acquired data on a BD X-20 or BD LSR II flow cytometer (BD Biosciences, San Jose, CA). We analyzed and visualized data with FlowJo v10.

Lin J.B., Sene A., Santeford A., Fujiwara H., Sidhu R., Ligon M.M., Shankar V.A., Ban N., Mysorekar I.U., Ory D.S, & Apte R.S. (2018). Oxysterol Signatures Distinguish Age-Related Macular Degeneration from Physiologic Aging. EBioMedicine, 32, 9-20.

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Flow Cytometric Analysis of Immune Cells

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Cells were washed in PBS and stained with Live/Dead Fixable Aqua stain (Invitrogen) for 30 min in the dark followed by washing with FACS wash buffer (PBS containing 05% FBS, 2 mM EDTA and 0.1% sodium azide). As a positive control for dead cell staining, 5×10⁵ cells were treated with heat at 65°C for 2 min, placed on ice for 2 min and mixed with equal number of live cells. Single-cell suspensions were incubated with TruStain FcX antibody (BioLegend) to block Fc receptors and then stained with directly conjugated antibodies (BD Bioscience; BioLegend). Flow cytometric analysis was performed on a FACSCanto (BD Biosciences) and 100,000 events were recorded for each sample. Data were analyzed using FACS Diva and FlowJo software (Tree Star). Gating was on Aqua Viability Stain negative CD45 positive populations, in which further markers were analyzed.

Wondimu A., Liu Y., Yan S., Bobb D., Ma J.S., Chakrabarti L., Radoja S, & Ladisch S. (2014). Gangliosides drive the tumor infiltration and function of myeloid-derived suppressor cells. Cancer research, 74(19), 5449-5457.

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Immunophenotyping T Lymphocyte Subsets

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For immunophenotyping T lymphocyte memory subsets, the TruStain FcX™ antibody (Clone 96, BioLegend) was added to block the non-specific binding of antibodies. Subsequently, splenocytes were incubated with Zombie RED for 10 min at room temperature. Finally, cells were stained with the following fluorophore-labeled anti-mouse monoclonal antibodies: anti-CD4 FITC (clone GK1.5, BioLegend), anti-CD8 PCy7 (clone QA17A07, BioLegend), anti-CD44 PE (clone IM7, BioLegend) and anti-CD62L PerCP (clone MEL-14, BioLegend) for 20 min at 4 °C in the dark. The T lymphocyte subsets were characterized by the percentage values obtained from the SLA-stimulated culture.

Hurtado-Morillas C., Martínez-Rodrigo A., Orden J.A., de Urbina-Fuentes L., Mas A, & Domínguez-Bernal G. (2024). Enhancing Control of Leishmania infantum Infection: A Multi-Epitope Nanovaccine for Durable T-Cell Immunity. Animals : an Open Access Journal from MDPI, 14(4), 605.

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Flow Cytometric Analysis of Murine Immune Cells

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Flow cytometry was performed using FACS Aria™ II (BD Biosciences, Franklin Lakes, NJ, United States). Samples were detached using 0.25% trypsin-EDTA and single cells were collected by filtration using a pluriStrainer Mini 40 µm (43-10040; pluriSelect, Leipzig, Germany). Cells were counted using a Countess^® II FL automated cell counter (Thermo Fisher Scientific, Waltham, MA, United States), following staining with Trypan blue. Single cells were stained in PBS containing 2% FBS, 2 mM EDTA (15575; Gibco), and TruStain FcX™ Antibody (101320; BioLegend, San Diego, CA, United States) for 30 min on ice using antibodies, followed by DAPI staining for dead cell detection. The following antibodies were used: PE anti-mouse Ly-6G antibody (127608; BioLegend), fluorescein isothiocyanate-conjugated anti-mouse CD45 antibody (103108; BioLegend), APC anti-mouse/human CD11b antibody (101212; BioLegend), PE rat IgG2a, κ isotype control antibody (400508; BioLegend), fluorescein isothiocyanate-conjugated rat IgG2b, κ isotype control antibody (400606; BioLegend), and APC rat IgG2b, κ isotype control antibody (400612; BioLegend). Data analyses were performed using FlowJo software (BD Biosciences).

Kase N., Kitagawa Y., Ikenaka A., Niwa A, & Saito M.K. (2023). A concise in vitro model for evaluating interactions between macrophage and skeletal muscle cells during muscle regeneration. Frontiers in Cell and Developmental Biology, 11, 1022081.

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Tumor Immune Profiling by Flow Cytometry

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Cao Y., Ye Q., Ma M, & She Q.B. (2023). Enhanced bypass of PD-L1 translation reduces the therapeutic response to mTOR kinase inhibitors. Cell reports, 42(7), 112764.

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Intracellular Cytokine Staining of Activated Splenocytes

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Upon stimulation and prior to harvest, isolated spleen cells were pre-incubated with brefeldin A (BioLegend, San Diego, CA, USA) for 4 h at 37 °C. To block non-specific antibody binding, the TruStain FcX™ antibody (Clone 96, BioLegend, San Diego, CA, USA) was then added. Subsequently, splenocytes were incubated with Zombie RED (Fixable Viability Kit, BioLegend, San Diego, CA, USA) for 10 min at room temperature, washed and immunostained with anti-CD4 FITC (clone GK1.5, BioLegend) (diluted 1:800) and anti-CD8 PE (clone QA17A07, BioLegend) (diluted 1:800) fluorophore-labeled antibodies. After two washes with PBS containing 2% FCS, cells were fixed and permeabilized using Cyto-FastTM Fix/Perm Solution (BioLegend) according to the manufacturer’s protocol. Then, cells were stained with anti-IFN-γ PerCP (clone XMG1.2, BioLegend) and anti-IL-10 PCy7 (clone JES5-16E3, BioLegend) at room temperature for 30 min, washed twice, and analyzed. The production of intracellular cytokines was expressed as percentages, representing the ratio of the percentage of IFN-γ or IL-10-producing-T cells in SLA-stimulated cultures.

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Macrophage Surface Marker Analysis

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Macrophages were incubated with TruStain FcX antibody (BioLegend, San Diego, CA, USA) for 10 min on ice to prevent unspecific binding of antibodies to Fc receptors. Then, cells were stained with rat anti-mouse CD86-APC (clone GL1, BioLegend, San Diego, CA, USA) or rat anti-mouse CD206-Brilliant Violet 421 (clone V068C2, BioLegend, San Diego, CA, USA) for 20 min at 4 °C in the dark and further washed with PBS supplemented with 2% FCS and 1 mM EDTA. Unstained cells were used as negative control for fluorescence. All data were collected by flow cytometry using FACSCanto II and DIVA software (Beckman Coulter, Brea, CA, USA).

Holthaus M., Santhakumar N., Wahlers T, & Paunel-Görgülü A. (2022). The Secretome of Preconditioned Mesenchymal Stem Cells Drives Polarization and Reprogramming of M2a Macrophages toward an IL-10-Producing Phenotype. International Journal of Molecular Sciences, 23(8), 4104.

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