Fc receptor blocking antibody

Manufactured by Thermo Fisher Scientific

The Fc receptor blocking antibody is a laboratory reagent that binds to Fc receptors, preventing their interaction with the Fc portion of antibodies. This allows for the specific detection or isolation of target cells or molecules without interference from Fc receptor-mediated effects.

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7 protocols using fc receptor blocking antibody

Multiparameter Analysis of iNKT and NK Cells

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All antibody staining of live cells was performed in Biolegend Cell Staining Buffer. Single cell suspensions were incubated in Fc-receptor blocking antibody (Clone 93, Thermo Fisher) on ice for 15 minutes prior to cell surface antigen staining. Dead cells were excluded with 7-aminoactinomycin D or Fixable Viability dyes (UV or eFluor 780, Thermo Fisher). For intracellular antigen staining, cells were fixed with either BD Pharmingen’s Cytofix/Cytoperm kit or Thermo Fisher’s FoxP3/Transcription Factor Fixation/Permeabilization kit for 30 minutes at room temperature. iNKT cells were identified as lymphocytes binding to anti-TCRβ antibodies and αGalCer analog PBS57-loaded CD1d tetramer (NIH Tetramer Core Facility/Emory Vaccine Center). For analysis of iNKT cells and NK cells, a “dump” channel with antibodies against CD19 and F4/80 was used to eliminate nonspecific staining. For neutral lipid staining, cells were stained with the LipidTox reagent (Thermo Fisher) per the manufacturer’s instructions. Gating strategies are provided in Supplemental Information (Figure S7).

LaMarche N.M., Kane H., Kohlgruber A.C., Dong H., Lynch L, & Brenner M.B. (2020). Distinct iNKT cell populations use IFNγ or ER stress-induced IL-10 to control adipose tissue homeostasis. Cell metabolism, 32(2), 243-258.e6.

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Optimizing ZPFCE-NP Labeling of BMDCs

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The in vitro labeling of BMDCs with ZPFCE-NPs was optimized at various concentrations (0, 1, 5 and 20 mM) and validated by flow cytometry. For the identification of ZPFCE-NP-labeled DCs, 2.5 × 10 5 BMDCs were first incubated with Fc receptor blocking antibody (Thermo Fisher Scientific, RRID:AB_467134) to minimize non-specific binding. In order to distinguish DCs from other immune cells and to evaluate the purity of the DC population, cells were surface stained with a cocktail of the following antibodies against major histocompatibility complex (MHC-II) I-A/I-E (Thermo Fisher Scientific, RRID:AB_465231) and CD11c (Thermo Fisher Scientific, RRID:AB_465553). Doublets were excluded based on signal height and width. Dead cells were stained by Zombie Yellow Fixable Viability Kit (Bio-Legend, 423,104, San Diego, CA, USA) according to the manufacturer's guidelines. They were excluded from analysis. Samples were read on a Gallios™ Flow Cytometer (Beckman Coulter, Analis, Suarlée, Belgium) and analyzed with the FlowJo software v10.1 (TreeStar, Ashland, OR, USA).

Saini S., Vanherwegen A.S., Liang S., Verbeke R., Korf H., Lentacker I., De Smedt S.C., Gysemans C, & Himmelreich U. (2022). Fluorine MR Imaging Probes Dynamic Migratory Profiles of Perfluorocarbon-Loaded Dendritic Cells After Streptozotocin-Induced Inflammation. Molecular imaging and biology, 24(2).

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FAP Expression Profiling of ASCs

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ASCs were harvested, resuspended in 2% FCS/PBS at 5x10⁵ cells/200μl, and transferred to a v-bottom 96 well plate. They were incubated 15 minutes with an Fc receptor blocking antibody (eBioscience 14-0161), 30 minutes with a biotinylated anti-FAP antibody (clone 73.3 available from EMD Millipore), and 15 minutes with streptavidin-conjugated brilliant violet 421 (Biolegend 405225), with two washes in between each incubation. Stained cells were finally resuspended in 2% FCS/PBS with propidium iodide and immediately run on a BD LSRFortessa cytometer.

Blomberg R., Beiting D.P., Wabitsch M, & Puré E. (2019). Fibroblast activation protein restrains adipogenic differentiation and regulates matrix-mediated mTOR signaling. Matrix biology : journal of the International Society for Matrix Biology, 83, 60-76.

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Lung Cell Isolation and Characterization

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Three mouse lungs from each treatment group were pooled and digested into single cell suspension with 0.4 mg/ml collagenase IV, followed by lysis of red blood cells. Each pooled treatment sample was separated into 3 technical replicates and incubated with Fc receptor-blocking antibody (eBioscience, San Diego, CA). Surface immunostaining of lung cell suspensions included CD31-PE (Invitrogen, Carlsbad, CA) for endothelial cells and CD45-APC (Invitrogen, Carlsbad, CA) for leukocytes, followed by staining with specific markers as below.

Fountain M.D., McLellan L.A., Smith N.L., Loughery B.F., Rakowski J.T., Tse H.Y, & Hillman G.G. (2019). Isoflavone-Mediated Radioprotection Involves Regulation of Early Endothelial Cell Death and Inflammatory Signaling in Radiation-Induced Lung Injury.. International journal of radiation biology, 96(2), 245-256.

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Eosinophil and Neutrophil Identification

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BALF and blood were processed immediately after collection and kept on ice throughout. After red blood cell lysis, cells were suspended in FACS buffer containing PBS, 2% FBS, 10 mM EDTA and 0.01% NaN₃. Suspensions were incubated with Fc receptor blocking antibody (eBioscience) to prevent non-specific binding. Cells were incubated on ice for 30 mins with a cocktail of antibodies to distinguish neutrophils from eosinophils. In mice, eosinophils were defined as CD45⁺CD11b⁺SiglecF⁺CD11c^loSSC^hi and neutrophils defined as CD45⁺CD11b⁺SiglecF^-CD11c^+/-Gr-1(Ly6G)^hi. Sample gating strategy is shown in S1 Fig. The following mouse antibodies were used: CD11c, CD11b, CD45 (Biolegend); Gr-1(Ly6G), SiglecF (BD Biosciences, San Jose CA), CD125 (R&D Systems). Channels with significant auto-fluorescence were avoided. After acquisition on a FACS Canto II, data analysis was performed using FlowJo 9.8.5 (BD, Ashland OR). Fluorescence minus one (FMO) controls were used to define gates. Data are expressed as percentage of CD125+ neutrophils to allow for longitudinal analysis of data collected at different timepoints, and as geometric mean fluorescence intensity (gMFI) for within-experiment comparisons.

Gorski S.A., Lawrence M.G., Hinkelman A., Spano M.M., Steinke J.W., Borish L., Teague W.G, & Braciale T.J. (2019). Expression of IL-5 receptor alpha by murine and human lung neutrophils. PLoS ONE, 14(8), e0221113.

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Characterizing Lung Immune Cell Responses Post-Irradiation

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BAL was performed prior to lung resection at 1, 8, 12, and 18 weeks after irradiation. Cells were loaded onto slides using a cytospin centrifuge and stained using a DiffQuik staining kit (IMEB Inc., San Marcos, CA,). Differential cell counts of leukocyte subsets were performed by counting at least 300 nucleated cells.²⁶Following collection of BAL fluids, the same mice provided the lungs for flow cytometry studies. Lungs were digested with 0.4 mg/mL collagenase IV and red blood cells were lysed. Lung single-cell suspensions were incubated with Fc receptor-blocking antibody (eBioscience, San Diego, CA) prior to staining. For morphological characterization of leukocytes, CD45⁺ cells were sorted by fluorescence-activated cell sorting (FACS) using a BD FACSVantage SE. CD45⁺ cell subsets were gated according to cell size and granularity. Cell subsets obtained from each gate were spun onto slides using a cytospin, and stained using a DiffQuik staining kit. To determine immunophenotype, cells were immunostained using a 5-color fluorophore combination of antibodies consisting of CD45-APC, CD11b-FITC, F4/80-PE, CD11c-APC-eFluor780, and Ly6G-PerCp-Cy5.5 (eBioscience). Fixable viability dye eFluor450 was used to exclude dead cells. Cells were analyzed by flow cytometry using a BD LSR II flow cytometer followed by analysis on FlowJo v10 software.

Abernathy L.M., Fountain M.D., Rothstein S.E., David J.M., Yunker C.K., Rakowski J., Lonardo F., Joiner M.C, & Hillman G.G. (2015). Soy Isoflavones Promote Radioprotection of Normal Lung Tissue by Inhibition of Radiation-Induced Activation of Macrophages and Neutrophils. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer, 10(12), 1703-1712.

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PbGAPDH and CD68 Binding Assays

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For binding assays of PbGAPDH or CD68 to macrophages or sporozoites, primary mouse peritoneal macrophages and Plasmodium sporozoites were harvested and fixed in 4% paraformaldehyde for 30 min. Mouse macrophages were initially treated with an Fc receptor–blocking antibody (eBioscience) for 10 min. For the PbGAPDH binding assay, mouse peritoneal macrophages were incubated in 5 mM PbGAPDH or pET32b tag protein (Fig. 5 D). For CD68 binding assay, P. berghei sporozoites were incubated in the culture supernatant of CD68-secreting 293T cells in presence of 18 mM PbGAPDH or pET32b tag protein (Fig. 5 E). Binding of PbGAPDH or CD68 was visualized with anti–His tag antibody (GenScript) and anti-CD68 antibody (AbD Serotec), respectively. Flow cytometry data were acquired with a FACSCalibur cell analyzer (BD) and analyzed using the FlowJo software (v8.7; Tree Star).
For immunofluorescence assays with antimimotope and control antibodies, sporozoites were fixed in 4% paraformaldehyde. For immunofluorescence assays with anti-PbGAPDH and control antibodies, sporozoites were incubated in Cytofix/Cytoperm solution (BD) per the manufacturer’s directions or in PBS as a control (Fig. 6 B). Alexa Fluor 488 (green)– or rhodamine-conjugated (red) secondary antibodies (Invitrogen) visualized sporozoites or peritoneal macrophages.

Cha S.J., Kim M.S., Pandey A, & Jacobs-Lorena M. (2016). Identification of GAPDH on the surface of Plasmodium sporozoites as a new candidate for targeting malaria liver invasion. The Journal of Experimental Medicine, 213(10), 2099-2112.

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