Fc block

Manufactured by BioXCell

Sourced in United States

The Fc Block is a laboratory reagent designed to prevent non-specific binding of antibodies to Fc receptors. It is a concentrated solution that can be added to samples or buffers to block Fc-mediated interactions, allowing for more accurate and specific immunoassays and other applications.

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Lab products found in correlation

Facscanto 2, by BD (5 mentions) Liberase, by Roche (2 mentions) Cd11b, by Thermo Fisher Scientific (2 mentions) F4 80, by Thermo Fisher Scientific (2 mentions) Disposable micropestles, by Corning (2 mentions) Cd11b clone m1 70, by BD (1 mentions) Cd11c clone hl3, by BD (1 mentions) Cd4 clone gk1, by BioLegend (1 mentions) Cd8α clone 53 6, by BioLegend (1 mentions) Cd19 clone 6d5, by BioLegend (1 mentions) Mhc class 2 clone m5 114, by BioLegend (1 mentions) Goat anti rabbit af488, by Thermo Fisher Scientific (1 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions) Live dead fixable violet stain, by Thermo Fisher Scientific (1 mentions) Lsrfortessa, by BD (1 mentions) Ab31704, by Abcam (1 mentions) Facs lysing solution, by BD (1 mentions) Facsaria, by BD (1 mentions) Lsr fortessa instrument, by BD (1 mentions) Ghost dye red 780, by Cytek Biosciences (1 mentions)

Spelling variants of this product

Fc block (2.4G2; (13 citations) Fc block (clone 2.4G2, (7 citations) Anti-CD16/CD32 Fc block (2 citations) Anti-CD16/32 Fc-block (2 citations) Fc Block (anti-CD16/32 clone 2.4G2; (2 citations)

11 protocols using fc block

Flow Cytometry Analysis of Immune Cell Subsets

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To detect surface proteins, mononuclear cells were incubated with Fc Block (Bio X Cell, 2.4G2) for 15 min and washed, followed by incubation with viability dye. The indicated antibodies were fluorescently conjugated against CD45 (clone 30-F11, BD Horizon, cat #563410), CD11b (clone M1/70, BD Biosciences, cat #563553), CD11c (clone HL3, BD Pharmingen, cat #553801), CD4 (clone GK1.5, BioLegend, cat #100428), CD8α (clone 53 – 6.7, BioLegend, cat #100734), CD19 (clone 6D5, BioLegend, cat #115541), and MHC Class II (clone M5/114.15.2, BioLegend, cat #107628). Samples were run on a BD Symphony (BD Biosciences) and analyzed using FlowJo software (Tree Star), as described (31 (link), 40 (link), 41 (link)).

Hong H., Wang Y., Menard M., Buckley J., Zhou L., Volpicelli-Daley L., Standaert D., Qin H, & Benveniste E. (2024). Suppression of the JAK/STAT Pathway Inhibits Neuroinflammation in the Line 61-PFF Mouse Model of Parkinson’s Disease. Research Square.

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Multiparameter Flow Cytometry Analysis

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Single-cell suspensions were incubated with Fc Block (Bio X Cell, 2.4G2) and stained with antibodies to surface markers diluted in FWB. Cells were washed in FWB and resuspended in FWB containing DAPI (Roche) and flow cytometric counting beads (CountBright Absolute; Life Technologies) for live cell gating and counts. Cells for intracellular staining were washed in PBS and stained with Violet Live/Dead fixable stain (Life Technologies) according to manufacturer’s instructions. Transcription factor staining was done using the FoxP3/Transcription Factor Staining Buffer Set (eBiosciences) according to manufacturer’s instructions. Cells for DCLK1 staining were fixed in 4% PFA for 2–5 min, washed in FWB, and stained with rabbit anti-DCLK1 (Abcam; ab31704; 1:4000) antibody in perm/wash buffer from the FoxP3 staining buffer kit, followed by F(ab’)2 donkey anti-rabbit IgG-PE (Life Technologies; 1:1000) or goat anti-Rabbit AF488 (Invitrogen; 1:4000) for 10 min. Staining was done on ice. For co-staining of DCLK1 and endogenous IL-25 RFP, PFA fixation was performed for 30 seconds on ice. Samples were analyzed on a LSRFortessa (BD Biosciences) with five lasers (355 nm, 405 nm, 488 nm, 561 nm and 640 nm). Samples were FSC-A/SSC-A gated to exclude debris, FSC-H/FSC-A gated to select single cells, and gated to exclude dead cells. Data were analyzed with FlowJo 9/10 (Treestar/Flowjo).

O’Leary C.E., Sbierski-Kind J., Kotas M.E., Wagner J.C., Liang H.E., Schroeder A.W., de Tenorio J.C., von Moltke J., Ricardo-Gonzalez R.R., Eckalbar W.L., Molofsky A., Schneider C, & Locksley R.M. (2022). Bile acid-sensitive tuft cells regulate biliary neutrophil influx. Science immunology, 7(69), eabj1080.

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Single-Cell Suspension Preparation Protocol

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Single cell suspensions were prepared from different organs. Prior to the preparation of single cell suspension organs were kept in 1x phosphate buffered saline (PBS) plus 2% (v/v) fetal calf serum (FCS). Ears were kept in only PBS. The spleens were isolated and filtered through a sterile sieve (40μm) to obtain single cell suspensions. Bones were flushed wit a syringe containing PBS plus 2% (v/v) FCS to extract cells from the bone marrow (BM). Red blood cells isolated from spleen and bone marrow, were lysed with tris-ammonium chloride, pH 7.2. Blood was lysed with BD FACS lysing solution (BD Pharmingen). Cells of the skin were digested with 0.25mg/ml liberase TM (Roche) and subsequently homogenized with 0.6mmx30mm needles. Single-cell suspensions were treated with Fc-Block (BioXCell) and surface stained with monoclonal antibodies: Ly6G (BL 1A8), CD90.2 (eBio 53–2.1), B220 (BL RA3-6B2), CD11b (eBio M1/70), F4/80 (eBio BM8), Ly6C (BD AL-21), Gr-1 (BD RB6-8C5), γδTCR (eBio eBioGL3), CD3e (BD 145-2C11), CD45.2 (BD 104), CD19 (BL 6D5, BD 1D3), CD126 (IL-6Rα) (D7715A7). For biotinylated antibodies streptavidin in V500 (BD Pharmingen) or Pe-Cy7 (eBio) was used. Dead cells were excluded with fixable viability dye ef780 (eBio). All samples were acquired with FACS Canto II (BD Pharmingen) and analyzed with FlowJo Version 8.87.

Klebow S., Hahn M., Nikoalev A., Wunderlich F.T., Hövelmeyer N., Karbach S.H, & Waisman A. (2016). IL-6 Signaling in Myelomonocytic Cells Is Not Crucial for the Development of IMQ-Induced Psoriasis. PLoS ONE, 11(3), e0151913.

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Single-cell Immune Profiling Protocol

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Single-cell preparations were incubated in PBS with 2% serum and Fc block (10 μg/ml; Bio X Cell) for 10 min on ice before staining with antibodies. Cells were stained for dead cells with Ghost Dye Red 780 (Tonbo Biosciences) for 15 min at room temperate in PBS. Surface marker was stained for 30 min at 4°C in PBS with 2% fetal bovine serum. For the intracellular cytokine staining, cells were stimulated in vitro with cell stimulation cocktail for 5 hours at 37°C with 5% CO₂ before staining. Next, cells were fixed and permeabilized with fixation and permeabilization buffer set (Tonbo Biosciences), after which they were stained with intracellular cytokine antibodies according to the manufacturer’s instructions. For intranuclear staining, the nuclear membranes were permeabilized with the Foxp3/Transcription Factor Staining Buffer Kit (eBioscience) before transcription factor staining. All analyses were performed on a LSRFortessa instrument (BD Biosciences). Data were processed using FlowJo software. Cell sorting was performed on a FACSAria (BD Biosciences). The antibodies and commercial assays are displayed in table S2.

Xie W., Fang J., Shan Z., Guo J., Liao Y., Zou Z., Wang J., Wen S., Yang L., Zhang Y., Lu H., Zhao H., Kuang D.M., Huang P., Chen Q, & Wang Z. (2022). Regulation of autoimmune disease progression by Pik3ip1 through metabolic reprogramming in T cells and therapeutic implications. Science Advances, 8(39), eabo4250.

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Multiparametric Flow Cytometry of Immune Cells

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Single cell solutions of the spleen, kidneys, aorta, and perivascular adipose tissue (PVAT) were prepared to be stained with fluorophore-coupled antibodies for flow cytometric analyses. After blocking unspecific binding with Fc-block (Bio X Cell, USA), the following monoclonal antibodies were used for surface staining: CD45.2 (clone: 104, APC-eFluor780, eBioscience), CD90.2 (clone: 53-2.1, APC-eFluor780, eBioscience or PerCP, BioLegend), CD3 (clone: 145-2C11, PerCP, BD Biosciences), CD4 (clone: GK1.5, FITC, BioLegend or clone: RM4-5, V500, BD Biosciences), CD8 (clone: 53-6.7, PE-Cy7, eBioscience), CD11b (clone: M1/70, PE-Cy7, eBioscience), F4/80 (clone: BM8, APC, BioLegend), Ly6G (clone: 1A8, PE, BioLegend), and Ly6C (clone: AL-21, BD Biosciences).
Samples were acquired using the FACSCanto™ II (BD). Analysis was performed using the FlowJo Software (BD, USA).

Schüler R., Efentakis P., Wild J., Lagrange J., Garlapati V., Molitor M., Kossmann S., Oelze M., Stamm P., Li H., Schäfer K., Münzel T., Daiber A., Waisman A., Wenzel P, & Karbach S.H. (2019). T Cell-Derived IL-17A Induces Vascular Dysfunction via Perivascular Fibrosis Formation and Dysregulation of ·NO/cGMP Signaling. Oxidative Medicine and Cellular Longevity, 2019, 6721531.

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Comprehensive Immune Cell Profiling

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Single cell suspensions were blocked with Fc-Block (BioXCell) in FACS buffer and surface stained with the following monoclonal antibodies: CD4 (Biolegend, RM4-5), CD8 (eBioscience, 53-6.7), CD19 (6D5, Biolegend), CD25 (BD, PC61), CD44 (eBioscience, IM7), CD62L (MEL-14, eBioscience), CD90.2 (eBioscience, 53–2.1), γδTCR (eBioscience, eBioGL3), Gr-1 (BD, RB6-8C5), F4/80 (eBioscience, BM8). To exclude dead cells, cells were also stained with the fixable viability dye ef780 (eBioscience).
For T_reg stainings, cells were fixed and permeabilized according to the PE anti-mouse/rat Foxp3 staining set (eBioscience) and intracellularly stained for Foxp3 (eBioscience, FJK-16s) and CTLA4 (BD, UC10-4F10-11). The cells were acquired with the FACS Canto II (BD Pharmingen) and analyzed with FlowJo Version 8.87. Cells were usually pregated for a lymphocyte gate, singlets and living cells.

Lacher S.M., Bruttger J., Kalt B., Berthelet J., Rajalingam K., Wörtge S, & Waisman A. (2017). HMG-CoA reductase promotes protein prenylation and therefore is indispensible for T-cell survival. Cell Death & Disease, 8(5), e2824-.

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Immunophenotyping Peripheral B Cells

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For flow cytometry of peripheral B cells, blood was collected in microtubes with EDTA to prevent coagulation and treated with ACK buffer (Lonza) to lyse red-blood cells. For lymph node samples, cell suspensions were obtained by mechanical disassociation with disposable micropestles (Axygen). Spleens were homogenized by filtering through a 70-μm cell strainer and treated with ACK buffer. Bone-marrow cells were extracted by centrifugation of punctured tibiae and femurs at up to 10,000 xG for 10 s, then treated with ACK buffer. Cells from each tissue were resuspended in PBS supplemented with 0.5% BSA and 1 mM EDTA and incubated first with FC-block (rat anti-mouse CD16/32, clone 2.4G2, Bio X Cell) for 30 min on ice and subsequently with various fluorescently-labeled antibodies (see Table S1) for 30 min. Cells were filtered and washed with the same buffer before analysis on a BD FACS Symphony cytometer. Data were analyzed using FlowJo v.10 software.

Petit R.J., Pineau E., Demesure B., Bacilieri R., Ducousso A, & Kremer A. (1997). Chloroplast DNA footprints of postglacial recolonization by oaks. Proceedings of the National Academy of Sciences of the United States of America, 94(18), 9996-10001.

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Multiparameter Flow Cytometry of Immune Cells

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Cell suspensions from spleens were prepared and filtered through a 40-μm cell strainer (Fisherbrand). Cells were washed and diluted in PBS with 1% FBS and were stained with Fc block (BioXCell) for 5 min, followed by surface staining for the indicated markers. Following labelled Abs were used: anti-CXCR5 (L138D7), anti-PD-1 (29F.1A12), anti-CD4 (RM4-5), anti-Foxp3 (MF-14), anti-CD38 (90) and anti-B220 (RA3-6B2) were obtained from BioLegend; GL7 (GL7) was purchased from BD Pharmingen. For intracellular staining, after surface markers were stained, cells were fixed and stained with anti-Foxp3, eBioscience intracellular kit was used. All samples were acquired on an LSR2 flow cytometer (Becton Dickonson) and analyzed with FlowJo software.

Xie M.M., Liu H., Corn C., Koh B.H., Kaplan M.H., Turner M.J, & Dent A.L. (2019). Roles of T follicular helper cells and T follicular regulatory cells in Autoantibody Production in IL-2-deficient mice. ImmunoHorizons, 3(7), 306-316.

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Lymph Node Cell Isolation and Sorting

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For flow cytometry and cell sorting, lymph node cell suspensions were obtained by mechanical disassociation with disposable micropestles (Axygen). Cells were resuspended in PBS supplemented with 0.5% BSA and 1 mM EDTA and incubated first with Fc-block (rat anti-mouse CD16/32, clone 2.4G2, Bio X Cell) for 30 min on ice and subsequently with various fluorescently-labeled antibodies (see Supplementary Table 1) for 30–60 min. Cells were filtered and washed with the same buffer before analysis on a BD FACS Symphony A5 cytometer or single-cell sorted using a BD FACS Symphony S6. Data were analyzed using FlowJo v.10 software.

Schiepers A., van ‘t Wout M.F., Hobbs A., Mesin L, & Victora G.D. (2023). Opposing effects of pre-existing antibody and memory T cell help on the dynamics of recall germinal centers. bioRxiv.

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Characterization of sEV Uptake in Mixed Glia

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Mixed culture glia cells were incubated with 5µL (containing about 2.6 × 10⁸ particles as measured with NTA) of WT or PrP-KO sEVs labelled with mCLING for 3 h. Cells were then trypsinized and transferred to FACS tubes containing FACS buffer (PBS with 1 mM EDTA and 0.2% BSA). Cells were centrifuged for 5 min at 4°C at 310xg, washed in FACS buffer and centrifuged again. Cells were stained for 30 min on ice with anti-CD11b–FITC (1:100; Clone M 1/70, Biolegend) and anti-GLAST-PE (1:100; MiltenyiBiotec) in the presence of Fc Block (1:100; Bio X Cell) in FACS buffer, washed two times with FACS buffer and finally resuspended in 200 µL of FACS buffer. Measurements were done with a BD FACSCanto™ II and analysed with FlowJo.

Brenna S., Altmeppen H.C., Mohammadi B., Rissiek B., Schlink F., Ludewig P., Krisp C., Schlüter H., Failla A.V., Schneider C., Glatzel M., Puig B, & Magnus T. (2020). Characterization of brain-derived extracellular vesicles reveals changes in cellular origin after stroke and enrichment of the prion protein with a potential role in cellular uptake. Journal of Extracellular Vesicles, 9(1), 1809065.

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