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Trustain fcx fc receptor blocking solution

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TruStain FcX Fc receptor blocking solution is a product designed to block Fc receptors in flow cytometry applications. It is intended to reduce non-specific binding of antibodies to Fc receptors, which can lead to false-positive results.

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Lab products found in correlation

Lsrii fortessa, by BD (1 mentions) Live dead fixable blue dead cell stain, by Thermo Fisher Scientific (1 mentions) Anti mouse cd16 32 clone 93, by BioLegend (1 mentions) Phorbol 12 13 dibutyrate, by Merck Group (1 mentions) Efluor 780, by Thermo Fisher Scientific (1 mentions) Brefeldin a, by Merck Group (1 mentions) Ionomycin, by Merck Group (1 mentions) Cytofix, by BD (1 mentions) Trustain fcx anti mouse cd16 32 ab, by BioLegend (1 mentions) Near ir amine dye, by Thermo Fisher Scientific (1 mentions) Cytofix cytoperm buffer, by BD (1 mentions) Saponin, by Merck Group (1 mentions) Cytofix fixation buffer, by BD (1 mentions) Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions) Nb100 2288, by Novus Biologicals (1 mentions) Anti o4 apc, by Miltenyi Biotec (1 mentions) Moflo astrios eq cell sorter, by Beckman Coulter (1 mentions) Anti mouse cd16 32 2.4g2, by BioLegend (1 mentions) Cytofix cytoperm kit, by BD (1 mentions) Lsrfortessa flow cytometer, by BD (1 mentions)

Spelling variants of this product

TruStain FcX (313 citations) Human TruStain FcX Fc receptor blocking solution (60 citations) Fc blocking solution (8 citations) TruStain FcX blocking solution (3 citations) TruStain FcX Receptor blocking solution (2 citations)

8 protocols using trustain fcx fc receptor blocking solution

1

Immunostaining and Flow Cytometry Analysis

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Cells were kept at 4 °C throughout immunostaining. First, cells were stained with LIVE/DEAD™ Fixable Blue Dead Cell Stain (ThermoFisher Scientific #L23105) at 1000x dilution for 30 min in PBS, after which staining was quenched with flow cytometry staining (FACs) buffer (Invitrogen #00-4222-26). Cells were blocked with TruStain FcX Fc receptor blocking solution (BioLegend #101319) for 5 min and stained with surface protein antibodies for 20 min (1:50 dilution), after which the cells were washed 3x in FACs buffer. Flow cytometry acquisition was performed on a BD Fortessa LSRII. Single color compensation beads (Thermo #01-2222-41), were used for multi-parameter flow cytometry compensation. Gating was done based on fluorescence-minus-one (FMO) controls. The complete set of antibodies used for flow cytometry is listed in Supplementary Table 3.
Adu-Berchie K., Brockman J.M., Liu Y., To T.W., Zhang D.K., Najibi A.J., Binenbaum Y., Stafford A., Dimitrakakis N., Sobral M.C., Dellacherie M.O, & Mooney D.J. (2023). Adoptive T cell transfer and host antigen-presenting cell recruitment with cryogel scaffolds promotes long-term protection against solid tumors. Nature Communications, 14, 3546.
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2

Multiparameter Flow Cytometry Analysis

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Cells were stained with the following antibodies: Brilliant Violet 421-conjugated anti-human CD45 (clone HI30), PE-Cy7 anti-mouse CD45 (30-F11), Alexa Fluor 700 anti-CD3 (HIT3a), FITC anti-CD4 (OKT4), PE-Dazzle594 anti-IL-4 (MP4-25D2), Alexa Fluor 647 anti-Foxp3 (259D), PerCP-Cy5.5 anti-CD127 (A019D5), PE-Cy7 anti-CD25 (BC96), PerCP-Cy5.5 anti-IFNγ (4S.B3), PE anti-IL-10 (JES3-19F1), APC anti-CD19 (HIB19), PE-Cy7 anti-HLA-DR (L243), Brilliant Violet anti-c-Kit (104D2), and PE anti-FcεRI (AER-37 (CRA-1)) were purchased from Biolegend. APC anti-IgE (Ige21) was obtained from Affymetrix eBioscience. Anti-mouse CD16/32 (clone 93) and TruStain FcX Fc receptor blocking solution (both Biolegend) were used to prevent non-specific binding. Dead cells were excluded using fixable viability dye eFluor 780 (Affymetrix eBioscience). Intracellular cytokine staining was performed after a 4hr stimulation at 37°C with 500ng/ml ionomycin, 500ng/ml phorbol 12,1 3-dibutyrate and 1µg/ml brefeldin A (all Sigma-Aldrich). Coordinate analysis of transcription factors and cytokine production was performed using BD Biosciences Cytofix and Cytoperm reagents as previously described6 (link). Intestinal leukocyte isolation was performed according to established protocols7 (link).
Burton O.T., Stranks A.J., Tamayo J.M., Koleoglou K.J., Schwartz L.B, & Oettgen H.C. (2016). A humanized mouse model of anaphylactic peanut allergy. The Journal of allergy and clinical immunology, 139(1), 314-322.e9.
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3

Multiparametric Flow Cytometry Protocol

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Non-specific binding of Abs was suppressed by incubation with human TruStain FcX™ Fc receptor blocking solution (BioLegend). For analysis of isolated tumor xenografts, cells were additionally incubated with TruStain fcX™ (anti-mouse CD16/32 Ab, BioLegend). 1 μg/mL DAPI was added to stain dead cells. Cells were labeled with the following Abs (clone) or MHC-peptide pentamers (HLA molecule, peptide sequence): CD3 (SK7), CD4 (OKT4), CD8α (HIT8a), CD45 (HI30), CD138 (DL-101), CD279/PD1 (EH12.2H7), CD318/CDCP1 (CUB1), HLA-A2 (BB7.2), HLA-A1/36 (8.L.104), HLA-A1/11/26 (8.L.101), pEBV_1 pentamer (HLA-A02:01, CLGGLLTMV), pEBV_2 pentamer (HLA-A02:01, GLCTLVAML), pFLU pentamer (HLA-A01:01, CTELKLSDY). Almost all Abs (including isotype controls) were purchased from BioLegend, except for the HLA-A1 Abs, which were obtained from Abcam. Pentamers were provided by ProImmune. Cells were analyzed using the BD Biosciences Canto II. Ab quality was validated in pilot experiments and gating was performed using isotype controls. The FlowJo (Treestar) software was used to analyze flow cytometry data and to calculate mean fluorescence intensity (MFI).
Sefrin J.P., Hillringhaus L., Mundigl O., Mann K., Ziegler-Landesberger D., Seul H., Tabares G., Knoblauch D., Leinenbach A., Friligou I., Dziadek S., Offringa R., Lifke V, & Lifke A. (2019). Sensitization of Tumors for Attack by Virus-Specific CD8+ T-Cells Through Antibody-Mediated Delivery of Immunogenic T-Cell Epitopes. Frontiers in Immunology, 10, 1962.
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4

Multiparameter Phenotyping of Cynomolgus PBMC

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Cynomolgus PBMC were stained with Near-IR amine dye (Life Technologies) to measure viability. The cells were washed with 2% FBS in PBS and blocked with TruStain FcX™ Fc Receptor blocking solution (BioLegend) for 15 min at 4°C. The cells were stained with an antibody cocktail containing non-human primate cross-reactive antibodies specific for CD4 (clone L200), CD8 (clone SK1), CD3 (clone SP34.2), CD20 (clone 2H7), CD16 (clone VEP13), HLA-DR (clone L243), CD38 (clone AT-1), PD-1 (clone EH12.1), CD25 (clone 4E3), CD28 (clone 28.8) and CD95 (clone DX95). Cells were fixed/permeabilized with Cytofix/Cytoperm buffer (BD Biosciences), as per manufacturer’s instructions. After washing, anti-Ki67 (clone B56) antibody was added and incubated for 15 min at 4°C. Cells were washed again with 2% FBS in PBS and the expression of Ki67 was evaluated by flow cytometry.
Gombos R.B., Gonzalez A., Manrique M., Chand D., Savitsky D., Morin B., Breous-Nystrom E., Dupont C., Ward R.A., Mundt C., Duckless B., Tang H., Findeis M.A., Schuster A., Waight J.D., Underwood D., Clarke C., Ritter G., Merghoub T., Schaer D., Wolchok J.D., van Dijk M., Buell J.S., Cuillerot J.M., Stein R., Drouin E.E, & Wilson N.S. (2018). Toxicological and pharmacological assessment of AGEN1884, a novel human IgG1 anti-CTLA-4 antibody. PLoS ONE, 13(4), e0191926.
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5

Fc Receptor Blockade and Viability Dye

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Cells were pre-incubated in PBS containing TruStain fcX™ Fc Receptor Blocking Solution (BioLegend) diluted 1:500, and eBioscience™ Fixable Viability Dye eFluor™ 780 diluted 1:1000 in order to block non-spiecific binding of immunoglobulins to the Fc receptors and exclude dead cells, respectively. Subsequently, cells were stained with specific anti-mouse antibodies for 45 min at 4°C. For intracellular staining of cytokines, the cells were fixated in BD Cytofix™ Fixation Buffer (BD Biosciences) prior to staining and 0.5% saponin (Sigma-Aldrich) was included in the flow cytometry buffer. After staining, cells were analyzed on FACSCanto II flow cytometer (BD Biosciences) and the data were analyzed and processed using FlowJo software.
Kuczkowska K., Copland A., Øverland L., Mathiesen G., Tran A.C., Paul M.J., Eijsink V.G, & Reljic R. (2019). Inactivated Lactobacillus plantarum Carrying a Surface-Displayed Ag85B-ESAT-6 Fusion Antigen as a Booster Vaccine Against Mycobacterium tuberculosis Infection. Frontiers in Immunology, 10, 1588.
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6

Circadian Protein Profiling by Flow Cytometry

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Cells were fixed and permeabilized with FIX and PERM (Nordic-MUbio, GAS-002). Thereafter, the samples were blocked with human TruStain FcX ™ Fc Receptor Blocking Solution (Biolegend, 422302), and circadian proteins were stained intracellular using primary antibodies NR1D1 (Abcam, ab174309), BMAL1 (Novusbio, NB100-2288), and ROR beta (Novusbio, NBP1-82532). The secondary donkey anti rabbit-PE (Biolegend, 406421) antibody was used for detection. Samples were measured by flow cytometry and analyzed as an increase over the isotype control.
Teppan J., Schwanzer J., Rittchen S., Bärnthaler T., Lindemann J., Nayak B., Reiter B., Luschnig P., Farzi A., Heinemann A, & Sturm E. (2024). The disrupted molecular circadian clock of monocytes and macrophages in allergic inflammation. Frontiers in immunology, 15.
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7

Fluorescence-Activated Cell Sorting Protocol

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Fluorescence activated cell sorting (FACS) was performed after differentiation. Adherent cultures were incubated with Accutase and gently dissociated. Cells were then spun down, resuspended in 50 μl DMEM/F12 + 0.5 μl TruStain FcX Fc Receptor Blocking Solution (BioLegend, San Diego, CA, United States), incubated at room temperature for 5 min, then immunolabeled with anti-O4-APC (Miltenyi, Gaithersburg, MD, United States, #130-119-155) for 45 min on ice. Live cells were then sorted with a MoFlo Astrios EQ cell sorter (Beckman-Coulter, Brea, CA, United States).
Plastini M.J., Desu H.L., Ascona M.C., Lang A.L., Saporta M.A, & Brambilla R. (2022). Transcriptional abnormalities in induced pluripotent stem cell-derived oligodendrocytes of individuals with primary progressive multiple sclerosis. Frontiers in Cellular Neuroscience, 16, 972144.
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8

Characterization of Immune Cell Populations

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Peripheral blood mononuclear cells from healthy donors were treated with TruStain FcX Fc Receptor Blocking Solution (BioLegend) and incubated with a mixture of fluorescently labeled antibodies against human CD3, CD4, CD8a, CD56, and CXCR3, or isotype control. In the mouse experiments, the cells were treated with anti‐mouse CD16/32 (2.4G2; BioLegend) to block the Fc receptors and incubated with a mixture of fluorescently labeled antibodies against mouse CD4, CD8a, CD45, CD49b, and CXCR3, or isotype control. For intracellular staining of mouse IFN‐γ, the cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences) and stained with the fluorescently labeled antibody. The antibodies used are listed in Table S1. The cells were analyzed using a BD LSRFortessa flow cytometer (BD Biosciences) and FlowJo software (Tree Star Inc.).
Higuchi T., Hashida Y., Matsuo K., Kitahata K., Ujihara T., Murakami I., Nakayama T, & Daibata M. (2023). EBV‐positive pyothorax‐associated lymphoma expresses CXCL9 and CXCL10 chemokines that attract cytotoxic lymphocytes via CXCR3. Cancer Science, 114(6), 2622-2633.
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