Fc block antibody

Manufactured by BD

Sourced in United States

Fc Block antibody is a laboratory reagent designed to block the Fc region of antibodies. It is used in various immunoassays and other applications to prevent non-specific binding and background signal.

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Fc block (765 citations) CD16/CD32 Fc Block antibody (4 citations) Anti-mouse CD16/CD32 Fc Block antibody (4 citations) CD16/32 antibody (Fc block; 2.4G2; (2 citations) Fc Block antibody (anti CD16/32, (2 citations)

13 protocols using fc block antibody

Quantifying Human Cell Engraftment

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Engraftment of human CD45⁺ cells and their subsets were determined by flow cytometry. In brief, cells were isolated from engrafted mice, blocked with human BD Fc block antibody (BD Biosciences, Cat#564219, RRID: AB_2728082) and mouse BD Fc block (2.4G2, BD Pharmingen, Cat#553141, RRID: AB_394656), and stained with combinations of antibodies purchased from Biolegend: Hematopoietic Stem and Progenitor Cell (HSPC) panel: APC/Cy7 mCD45 (30-F11, 1:300, Cat#103116, RRID: AB_312981), APC/Cy7 mTer119 (Ter-119, 1:300, Cat#116223, RRID: AB_2137788), BV510 hCD45 (HI30, 1:100, Cat#304036, RRID: AB_2561940), BV421 huCD38 (HIT2, 1:100, Cat#303526, RRID: AB_10983072), PE huCD34 (561, 1:100, Cat#343606, RRID:AB_1732008), PE/Cy7 huCD10 (HI10a, 1:100, Cat#312214, RRID: AB_2146548). Human cell engraftment panel: APC/Cy7 mCD45 (30-F11, 1:300), APC/Cy7 mTer119 (Ter-119, 1:300), BV510 hCD45 (HI30, 1:100), FITC huCD3 (OKT3, 1:100, Cat#317306, RRID: AB_571907), PE/Cy7 huCD19 (HIB19, 1:100, Cat#302216, RRID: AB_314246), APC huCD33 (WM53, 1:100, Cat#983902, RRID: AB_2810824), PE huCD34 (561, 1:100). Data were acquired with FACSDiva on an LSR Fortessa (BD Biosciences) equipped with 5 lasers and analyzed with FlowJo V10 software.

Gbyli R., Song Y., Liu W., Gao Y., Biancon G., Chandhok N.S., Wang X., Fu X., Patel A., Sundaram R., Tebaldi T., Mamillapalli P., Zeidan A.M., Flavell R.A., Prebet T., Bindra R.S, & Halene S. (2022). In Vivo Anti-Tumor Effect of PARP Inhibition in IDH1/2 Mutant MDS/AML Resistant to Targeted Inhibitors of Mutant IDH1/2. Leukemia, 36(5), 1313-1323.

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T Cell Immune Response Profiling

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T cells were isolated from the spleens of immunized C57BL/6 mice or APP₆₉₅ transgenic mice on day 7 after the fourth immunization. For intracellular staining, T cells were stimulated with Aβ42 protein at 10 μg/ml for 8 hours and subsequently treated with brefeldin A (BFA; BD Biosciences, San Diego, CA, USA) for 2 hours in vitro. The cells were blocked with BD Fc Block antibody (BD Biosciences) in phosphate-buffered saline (PBS) for 30 minutes at 4°C before being fixed with 4% paraformaldehyde and permeabilized with saponin (Sigma-Aldrich). The splenocytes were intracellularly stained with the appropriate concentrations of phycoerythrin (PE)-labeled antibodies, including anti-Foxp3, interleukin 10 (anti-IL-10), transforming growth factor β (anti-TGF-β), interferon γ (anti-IFN-γ), anti-IL-4 and PE-cyanine 5.5 (PE-Cy5.5)-labeled anti-CD25 antibodies (eBioscience, San Diego, CA, USA) and fluorescein isothiocyanate (FITC)-labeled anti-CD4 antibody (eBioscience) for 30 minutes at 4°C. The cells were washed and analyzed with a FACSCalibur flow cytometer equipped with CellQuest Pro software (BD Biosciences).

Wang S., Yu Y., Geng S., Wang D., Zhang L., Xie X., Wu B., Li C., Xu H., Li X., Hu Y., Zhang L., Kaether C, & Wang B. (2014). A coimmunization vaccine of Aβ42 ameliorates cognitive deficits without brain inflammation in an Alzheimer’s disease model. Alzheimer's Research & Therapy, 6(3), 26.

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Quantifying Human Cell Engraftment

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Prostate Cell Immunophenotyping by Flow Cytometry

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Dissociated prostate cells were suspended in DME-10% FBS, preincubated with purified rat anti-mouse CD16/CD32 (mouse BD Fc Block) antibody (1:50, BD Pharmingen, San Jose, CA) at 4°C for 10 min, and stained with specific antibodies at 4°C for 30 min. Flow cytometry analysis was performed using a BD FACS Canto II and analysed by NovoExpress^™ software (ACEA Biosciences, San Diego, CA). Cell sorting was conducted on a BD FACS Vantage or a BD FACS Aria. Primary antibodies used were: PE-Cy5 rat anti-human/mouse CD49f (1:100, BD Pharmingen), APC rat anti-human/mouse CD49f (1:100, BD Pharmingen), APC rat anti-mouse CD24 (1:100, BD Pharmingen), PE rat anti-mouse Sca-1 (1:100, eBioscience, San Diego, CA). Isotype controls used were from BD Pharmingen : PE rat IgG (1:100), PE-Cy5 Rat IgG (1:100), APC Rat IgG (1 :100). For lineage staining, biotinylated antibodies used were : rat anti-mouse CD31 (1:100, BD Pharmingen), rat anti-mouse CD45 (1:100, eBioscience), rat anti-mouse TER-119 (1:100, eBioscience). Dynabeads M-280 Streptavidin (Invitrogen) or BD Horizon™ V450-Streptavidin (1 :10000, BD Biosciences, Franklin Lakes, NJ) were used.

Brocqueville G., Chmelar R.S., Bauderlique-Le Roy H., Deruy E., Tian L., Vessella R.L., Greenberg N.M., Rohrschneider L.R, & Bourette R.P. (2016). s-SHIP expression identifies a subset of murine basal prostate cells as neonatal stem cells. Oncotarget, 7(20), 29228-29244.

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Lung Immune Cell Profiling by Flow Cytometry

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Mice were perfused with sterile PBS and left lobes of lungs were digested at 37°C with 630 µg/ml collagenase D (Roche) and 75 U/ml DNase I (Sigma). All antibodies were used at a dilution of 1:200. Single cell suspensions were preincubated with Fc Block antibody (BD PharMingen) in PBS + 2% heat-inactivated FBS for 10 min at room temperature before staining. Cells were incubated with antibodies against the following markers: V500 anti-CD45.1 (clone A20), AF700 anti-CD45.2 (clone 104; eBioscience), AF700 anti-CD45 (clone 30 F-11), APC-Cy7 anti-CD11c (clone N418), PE anti-Siglec F (clone E50-2440; BD), PE-Cy7 anti-Ly6G (clone 1A8), PB or Qdot605 anti-Ly6C (clone AL-21/HK1.4; Biolegend/BD), PerCP-Cy5.5 anti-CD11b (clone M1/70), APC anti-CD103 (clone 2E7; eBioscience), PB anti-CD3 (clone 17A2), PE-Cy7, APC anti-CD4 (clone RM4-5), PE-Cy7 anti-CD8 (clone53-6.7), anti-NK1.1 (clone PK136), and APC anti-Nos2 (clone CXNFT; all from eBioscience). Intracellular ROS were stained with MitoSOX red dye (Thermo Fisher). Absolute cell counts were determined using TruCount beads (BD). Cells were stained for 20 min at 4°C, washed, and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 20 min at 4°C. Flow cytometry data were acquired on a cytometer (LSR Fortessa; BD Biosciences) and analyzed using FlowJo software (Tree Star). Gating strategies are depicted in Fig. S2.

Nair S., Huynh J.P., Lampropoulou V., Loginicheva E., Esaulova E., Gounder A.P., Boon A.C., Schwarzkopf E.A., Bradstreet T.R., Edelson B.T., Artyomov M.N., Stallings C.L, & Diamond M.S. (2018). Irg1 expression in myeloid cells prevents immunopathology during M. tuberculosis infection. The Journal of Experimental Medicine, 215(4), 1035-1045.

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Dissociation and FACS Sorting of Retinal Cells

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The dissected retinas were dissociated in EBSS containing papain and DNase (Worthington) for 20 min at 37°C, with gentle shaking every 5 min, then digestion was stopped by adding Ovomucoid/EBSS (Worthington). The dissociated retinal cells were incubated with the FC block antibody (1:100, BD Biosciences 553141), and then stained with Thy1.2-PE antibody (1:100, Invitrogen 12-0902-82) and DAPI. Fluorescence-activated cell sorting (FACS) sorting was performed with a BD FACSAria IIIu instrument.

Peng X.Q., Dai S.K., Li C.P., Liu P.P., Wang Z.M., Du H.Z., Teng Z.Q., Yang S.G, & Liu C.M. (2020). Loss of Arid1a Promotes Neuronal Survival Following Optic Nerve Injury. Frontiers in Cellular Neuroscience, 14, 131.

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Quantitative Analysis of Puromycin-Induced Mitochondrial Changes

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Cells were infected as described above and 15 min before the end of the experiment media was supplemented with 91 μM puromycin and 208 μM emetine. One well was left without puromycin and used as negative control (C−). Cells fixed with 3% paraformaldehyde, protease inhibitors cocktail cOmplete Mini (Roche), 0.015% digitonin and 10 U /mL of RNaseOUT in 50mM Tris-HCl buffer for 15 min at room temperature, blocked in a solution of 0.05% saponin, 1 μg/mL of Fc block antibody (BD Biosciences), 10 mM glycine and 5% FBS in PBS for 10 min, followed by addition of 1:100 of 2D10-conjugated to Alexa 488 or Alexa 647 fluorophores, a kind gift of Jonathan W Yewdell (NIAID). Cells were counterstained with Hoechst at 0.1μg/mL and fluorescence quantified in iCys Compucyte LSC. For mitochondrial morphology cells were plated in optical quality dishes (Maktek Corporation), stained for RPM as before or with Alexa 488 conjugated-cytochrome c antibody, clone 6H2.B4 (BDPharmingen) and imaged 0.2 μm steps with Inverted Olympus IX71 coupled to a Photometrics CoolSnap HQ CCD camera and analyzed using Volocity 3D (Perkin Elmer).

Coelho C., Souza A.C., Derengowski L.D., de Leon-Rodriguez C., Wang B., Leon-Rivera R., Bocca A.L., Gonçalves T, & Casadevall A. (2015). Macrophage mitochondrial and stress response to ingestion of Cryptococcus neoformans. Journal of immunology (Baltimore, Md. : 1950), 194(5), 2345-2357.

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Flow Cytometry Immune Cell Profiling

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Cells were resuspended and blocked with Fc block antibody (BD, Biosciences) for 20 min at room temperature. Then, antibody mix was added to the cells and stained for 30 min at room temperature in the dark. All samples were analyzed by using an LSRFortessa flow cytometer (BD Biosciences). Fluorochrome-conjugated mAbs specific for CD45, CD11b, CD86, Ly6G (1A8), and CD206 (2B10C42) were purchased from BioLegend. Doublets were excluded by gating out the population defined by forward scatter and side scatter in the flow cytometry in all the experiments.

Zhang G., Li Q., Tao W., Qin P., Chen J., Yang H., Chen J., Liu H., Dai Q, & Zhen X. (2023). Sigma-1 receptor-regulated efferocytosis by infiltrating circulating macrophages/microglial cells protects against neuronal impairments and promotes functional recovery in cerebral ischemic stroke. Theranostics, 13(2), 543-559.

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Immune Cell Depletion Analysis Protocol

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For analysis of immune cell depletion, peripheral blood cells were collected, and erythrocytes were lysed with ACK lysis buffer (Gibco) and resuspended in RPMI supplemented with 10% FBS. Single cell suspensions were preincubated with Fc Block antibody (BD PharMingen) in PBS with 2% FBS for 10 min at room temperature before staining. Cells were incubated with antibodies against the following markers: BV421 anti-CD45, AF700 anti-Ly6C, FITC anti-Ly6B, PE-CY7 anti-Ly6G and APC anti-CD11b. All antibodies were used at a dilution of 1:200. Cells were stained for 20 min at 4°C, washed with PBS, fixed with 4% PFA for 15 min, washed with PBS and resuspended with FACS (PBS, 2% FBS, and 2 mM EDTA) buffer. For the lungs, single cells suspensions were collected as described below and stained in the same way as peripheral blood.

Chong Z., Karl C.E., Halfmann P.J., Kawaoka Y., Winkler E.S., Keeler S.P., Holtzman M.J., Yu J, & Diamond M.S. (2022). Nasally delivered interferon-λ protects mice against infection by SARS-CoV-2 variants including Omicron. Cell Reports, 39(6), 110799.

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Multiparametric Flow Cytometry of Lung Immune Cells

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Cells collected from BAL were stained with mAbs specific for CD45, CD3, CD8, CD4, CD44, CD11b, CD11c, Ly6G, Ly6C, MHC-II, γδ-TCR, F4/80, and B220 to define myeloid and lymphoid populations. Refer to Key Resources Table (S2 Table) for full panel of antibodies used in immune cell characterization. Gating strategies are depicted in Fig 2A.
For analysis of cells that produce IL-12p40, whole lungs from H5N1-VN/PR8 infected IL12b-yet40 or WT mice were digested at 37°C with 500μg/mL collagenase D (Sigma; #C-0130), 10μg/mL DNase I (Sigma; #D-4263), and 10mM HEPES in HBSS media. Single cell suspensions were pre-incubated with Fc Block antibody (BD Pharmingen) in PBS/2% FBS for 10 min at 4°C before staining. All antibodies were used at a dilution of 1/200. Cells were stained for 30 min at 4°C, washed, and fixed in 2% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 min at 4°C. All flow analysis samples were run on a CantoII flow cytometer (BD Biosciences). The resulting data was analyzed using FlowJo software (Treestar).

Gounder A.P., Yokoyama C.C., Jarjour N.N., Bricker T.L., Edelson B.T, & Boon A.C. (2018). Interferon induced protein 35 exacerbates H5N1 influenza disease through the expression of IL-12p40 homodimer. PLoS Pathogens, 14(4), e1007001.

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