Fc block 2.4g2

Manufactured by BioXCell

Sourced in United States

The Fc block (2.4G2) is a laboratory reagent designed to block Fc receptors. It functions by binding to Fc receptors on the surface of cells, preventing the binding of antibodies that may interfere with downstream applications.

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Lab products found in correlation

Fortessa cytometer, by BD (3 mentions) Mouse regulatory t cell staining kit, by Thermo Fisher Scientific (3 mentions) Bv421, by BioLegend (2 mentions) Facscanto 2, by Tree Star (2 mentions) Pe conjugated anti ctla 4 uc10 4f10 11 pe cy7 conjugated anti cd25 pc81 biotin conjugated anti cd8b 53 5.8, by BioLegend (2 mentions) Pacific blue conjugated anti cd4 rm4 5, by BioLegend (2 mentions) Percp cy5.5 conjugated anti thy1 1 ox 7, by Thermo Fisher Scientific (2 mentions) Brilliant violet421 conjugated anti human cd4 okt4, by Thermo Fisher Scientific (2 mentions) Apc conjugated il 10 jess 16e3, by Cytek Biosciences (2 mentions) Efluor660 conjugated anti gata 3 twaj, by Cytek Biosciences (2 mentions) Fitc conjugated anti cd4 gk1.5, by Thermo Fisher Scientific (2 mentions) R pe conjugated anti human cd4 s3.5, by Thermo Fisher Scientific (2 mentions) Facs aria fusion, by Tree Star (2 mentions) Foxp3 staining buffer set, by Thermo Fisher Scientific (2 mentions) Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions) Facscanto 2, by BD (2 mentions) Efluor 780, by Thermo Fisher Scientific (2 mentions) Facscelesta, by BD (2 mentions) α cxcr5 2g 8, by BD (2 mentions) α cd4 rm4 5, by BD (2 mentions)

13 protocols using fc block 2.4g2

Evaluating CAR T Cell Specificity

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To evaluate CAR specificity, cells were stained with various peptide:IA^g7 tetramers. Soluble peptide:IA^g7 proteins were produced using S2 insect cells as previously described and made into tetramers by conjugating the soluble peptide:IA^g7 molecules with BV421 (BioLegend), PE, or APC (Prozyme/Agilent) streptavidin at a 4.5:1 ratio (63 (link)). The tetramers used were InsBP8E:IA^g7, InsBP8G:IA^g7, p63:IA^g7, and HEL_11–25:IA^g7.
For in vivo experiments, single-cell suspensions were obtained from spleen and LNs by mechanical disruption. Lymphocytes were isolated from pancreas by collagenase P and DNase digestion, followed by Percoll density gradient centrifugation at 800 g for 20 minutes. Single-cell suspensions were stained with InsBP8E:IA^g7 tetramers, antibodies against CD4, CD8a, CD11c, CD11b, F4/80, CD90.1, CD90.2, CD45.1, CD45.2, and PD-1, and fixable live/dead dye for 30 minutes at 4°C in the presence of Fc block (2.4G2; Bio X Cell), fixed and permeabilized (Tonbo Biosciences), and stained for intracellular antigens FOXP3, HELIOS, IFN-γ, TNF-α, CTLA-4, and Ki67 overnight at 4°C in permeabilization buffer. A complete list of antibodies can be found in Supplemental Table 1. Flow cytometry was performed on LSRII or Fortessa cytometers (BD Biosciences) and analyzed using FlowJo software, version 10.8 (BD Biosciences).

Spanier J.A., Fung V., Wardell C.M., Alkhatib M.H., Chen Y., Swanson L.A., Dwyer A.J., Weno M.E., Silva N., Mitchell J.S., Orban P.C., Mojibian M., Verchere C.B., Fife B.T, & Levings M.K. (2023). Tregs with an MHC class II peptide–specific chimeric antigen receptor prevent autoimmune diabetes in mice. The Journal of Clinical Investigation, 133(18), e168601.

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Multiparametric Flow Cytometry Profiling

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Cells were stained at 4 °C in the presence of Fc Block (2.4G2; BioXcell) in flow cytometry buffer (0.5% BSA in PBS). The following antibodies were purchased from Becton Dickinson (BD): PE-conjugated anti-CTLA-4 (UC10-4F10-11); PE-Cy7–conjugated anti-CD25 (PC81); biotin-conjugated anti-CD8b (53-5.8), from BioLegend: Pacific blue-conjugated anti-CD4 (RM4-5); PerCP/Cy5.5–conjugated anti-Thy1.1 (OX-7); Brilliant Violet421–conjugated anti-human CD4 (OKT4); APC-conjugated anti-human CD4 (RPA-T4), biotin-conjugated anti CD45R/B220 (RA3-6B2), from eBioscience: APC-conjugated IL-10 (JESS-16E3); efluor660-conjugated anti-GATA-3 (TWAJ); PE-conjugated anti-IRF4 (3E4), biotin-conjugated anti-CD49b (DX5), from Tonbo Biosciences: FITC conjugated anti-CD4 (GK1.5), from Invitrogen, R-PE-conjugated anti-human CD4 (S3.5). For IL-10 and CTLA-4 staining, cells were fixed in 2% paraformaldehyde for 15 min at RT and permeabilized with 0.5% Saponin before staining. For BATF, GATA-3 and IRF4, cells were fixed and permeabilized with Foxp3 Staining Buffer Set (eBioscience) following manufacturers’ instructions. Cells were analyzed on a FACSCanto II or FACS Aria Fusion and data were analyzed with FlowJo software (TreeStar).

Iwata A., Durai V., Tussiwand R., Briseño C.G., Wu X., Grajales-Reyes G.E., Egawa T., Murphy T.L, & Murphy K.M. (2017). Quality of TCR signaling encoded by differential enhancer affinities for the composite BATF-IRF4 transcription factor complex. Nature immunology, 18(5), 563-572.

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Insulin-reactive T and B cell profiling

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Single cell suspensions were stained with PE- and APC-conjugated insB_10-23:I-A^g7 tetramers (p8E, p8G) and anti-CXCR5 for 1 hour at 25C in the presence of Fc block (2.4G2; BioXCell) followed by 30 minute incubation at 4C with cell surface marker antibodies, fixed and permeabilized (Tonbo Biosciences), and stained intracellularly (Supplemental Table 1) [28 (link)]. Antibodies, clones, and colors listed in Supplemental Table 1 are from BD Biosciences (San Jose, CA), BioLegend, Tonbo, and Thermo Fisher Scientific. Insulin-specific CD4⁺ T cells were defined as: singlets, live, lineage^neg (B220, CD8, CD11b, CD11c), CD4⁺, PE- and APC-tetramer⁺ cells. To detect B cells, samples were stained with 10nM decoy reagents for 10min at 25C followed by 10nM tetramers for 30min at 4C [19 (link)]. Tetramer⁺ cells were magnetically enriched from spleen and other lymph node cell suspensions (EasySep, STEMCELL Technologies), surface stained, fixed, permeabilized and stained with anti-Ig(H+L) (Supplemental Table 1). Insulin-reactive B cells were defined as: singlets, live, lineage^neg (CD11c, F4/80, Gr1, CD90.2), decoy^neg, PE- and APC-tetramer⁺ cells. Flow cytometry was performed on BD Biosciences LSRII or Fortessa cytometers and analyzed using FlowJo software (FlowJo, Ashland, OR; BD Biosciences).

Martinov T., Swanson L.A., Breed E.R., Tucker C.G., Dwyer A.J., Johnson J.K., Mitchell J.S., Sahli N.L., Wilson J.C., Singh L.M., Hogquist K.A., Spanier J.A, & Fife B.T. (2019). Programmed death-1 restrains the germinal center in type 1 diabetes. Journal of immunology (Baltimore, Md. : 1950), 203(4), 844-852.

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Isolation and Analysis of Lung-Draining CD8+ T Cells

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Cell suspensions from lung-draining, mediastinal lymph nodes (mLNs) were harvested, mechanically disrupted and filtered through a 70 µm nylon cell strainer (BD Biosciences). Cells were washed and resuspended in PBS with 2% donor calf serum and 10 µg/ml FcBlock (2.4G2 -BioXCell) for 10 min on ice before staining with fluorochrome-conjugated antibodies. Fluorochrome-labeled anti-CD8 (53-6.7), anti-CD19 (1D3), anti-CD45.1 (A20), CD45.2 (104) anti-CD25 (PC61), anti-Bcl6 (clone K112.91, dilution 1/50), anti-CXCR5 (clone 2G-8, dilution 1/50), anti-CD4 (clone RM4–5), anti-Blimp-1 (clone 5E7) were from BD Biosciences. Anti-T-bet (clone 4B10) was purchased from Biolegend. Dead cell exclusion was performed using 7-AAD (Calbiochem). The H-2D^b class I tetramer containing NP_366–374 peptide (NP) was generated by the NIH Tetramer Core Facility (Atlanta, GA). When indicated, the intracellular staining was performed using the mouse regulatory T cell staining kit (eBioscience) following manufacturer’s instructions. Flow cytometry was performed using a FACSCanto II (BD Biosciences) and an Attune NxT Flow Cytometer (ThermoFischer Scientific). In the indicated experiments, donor CD25^hi and CD25^lo OTI cells and endogenous CD25^hi and CD25^lo NP-specific CD8⁺ T cells were sorted using a FACSAria II sorter (BD Biosciences).

Chisolm D.A., Savic D., Moore A.J., Ballesteros-Tato A., León B., Crossman D.K., Murre C., Myers R.M, & Weinmann A.S. (2017). CCCTC-Binding Factor Translates Interleukin 2- and α-Ketoglutarate-Sensitive Metabolic Changes in T Cells into Context-Dependent Gene Programs. Immunity, 47(2), 251-267.e7.

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Lymph Node and Spleen Dissociation

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Mesenteric and peripheral lymph nodes and spleens were collected from each mouse, mechanically dissociated, and then enzymatically disaggregated as described previously [20] . Samples were centrifuged at 1600 RPM at +4°C and resuspended to a final volume of 200 µl sorter buffer (DPBS + 2% fetal bovine serum, 0.1% sodium azide) including Fc block (2.4G2, BioXCell, West Lebanon, NH).

Pravetoni M., Vervacke J.S., Distefano M.D., Tucker A.M., Laudenbach M, & Pentel P.R. (2014). Effect of Currently Approved Carriers and Adjuvants on the Pre-Clinical Efficacy of a Conjugate Vaccine against Oxycodone in Mice and Rats. PLoS ONE, 9(5), e96547.

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Comprehensive Immune Cell Profiling

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Antibodies against the following proteins were purchased from BD Biosciences: CD4 (RM4-5), CD69 (H1.2F3), CD90.1 (OX-7), phospho-STAT5^Y694 (C71E5), and CD90.2 (53-2.1). Antibodies against the following proteins were purchased from eBioscience: CD44 (IM7), CD98 (RL388), ICOS (7E.17G9), IRF4 (3E4), Ki-67 (SolA15), CD39 (24DMS1), KLRG1 (2F1), and FoxP3 (FJK-16s). Antibodies against the following proteins were purchased from BioLegend: CD4 (RM4-5), CD45 (30-F11), CD62L (MEL-14), CTLA-4 (UC10-4F10-11), PD-1 (29F.1A12), CD25 (PC61), and Bcl2 (BCL/10C4). Normal rabbit IgG (2729) and anti-phospho-S6^S240/244 (5364) were purchased from Cell Signaling Technology. Goat anti-rabbit-Alexa Fluor 647 secondary antibody was purchased from Invitrogen. Fc Block (2.4G2) and anti-CD28 (37.51) were purchased from Bio X Cell. Stimulatory anti-CD3 (2C11) was purified from hybridoma supernatants prepared in-house. Fixable viability dye eFluor780 was purchased from eBioscience. MitoTracker Deep Red dye was purchased from Invitrogen. Flow cytometry experiments were performed on a FACSCalibur, LSR II, or FACSCelesta (BD Biosciences), and analyzed using FlowJo software (Treestar, v.10.3) or FCS Express (De Novo Software, v.6). Cell sorting was performed on a FACSAria II or FACSAria Fusion (BD Biosciences).

Sun I.H., Oh M.H., Zhao L., C.h.i.r.a.g., Patel H., Arwood M.L., Xu W., Tam A.J., Blosser R.L., Wen J, & Powell J.D. (2018). mTORC1 signaling regulates the generation and function of central and effector FoxP3+ regulatory T cells : mTORC1 signaling controls central and effector Tregs. Journal of immunology (Baltimore, Md. : 1950), 201(2), 481-492.

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Flow Cytometry of Adipose Tissue Macrophages

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Flow cytometry data were acquired on an LSR-II flow cytometer (Becton Dickinson) and analyzed using FlowJo software (TreeStar 8.7) and Cytobank (https://www.cytobank.org) for dimensionality reduction and visualization by t-SNE. All stainings were performed as recommended by the antibody manufacturer. All samples were preincubated with Fc block (2.4G2; Bioxcell; BE0307) for 15 min at 4°C. FACS sorting was performed using an ARIA II sorter using a 100-µm nozzle (Becton Dickinson). Flow cytometry of ATMs is made somewhat difficult because of their high autofluorescence. To minimize the impact of autofluorescence, we meticulously selected the fluorochromes for each cell marker and used FMOs.

Silva H.M., Báfica A., Rodrigues-Luiz G.F., Chi J., Santos P.D., Reis B.S., Hoytema van Konijnenburg D.P., Crane A., Arifa R.D., Martin P., Mendes D.A., Mansur D.S., Torres V.J., Cadwell K., Cohen P., Mucida D, & Lafaille J.J. (2019). Vasculature-associated fat macrophages readily adapt to inflammatory and metabolic challenges. The Journal of Experimental Medicine, 216(4), 786-806.

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CAR Specificity Assessment via Peptide:IA^g7 Tetramers

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To evaluate CAR specificity, cell were stained with various peptide:IA^g7 tetramers. Soluble peptide:IA^g7 proteins were produced using S2 insect cells as previously described and made into tetramers by conjugating the soluble peptide:IA^g7 molecules with BV421 (BioLegend), PE, or APC (Prozyme/Agilent) streptavidin at a 4.5:1 ratio (59 (link)). The tetramers used were Insulin B10–23P8E:IA^g7, p63:IA^g7, and HEL_11–25:IA^g7.
For in vivo experiments, single-cell suspensions were obtained from spleen and lymph nodes by mechanical disruption. Lymphocytes were isolated from pancreas by collagenase P and DNase digestion, followed by Percoll density gradient centrifugation. Single cell suspensions were stained with InsBP8E:IA^g7 tetramers, antibodies against CD4, CD8a, CD11c, CD11b, F4/80, CD90.1, CD90.2, CD45.1, CD45.2, PD-1, and fixable live/dead dye for 30min at 4°C in the presence of Fc block (2.4G2; Bio X Cell), fixed and permeabilized (Tonbo Biosciences, San Diego, CA), and stained for intracellular antigens Foxp3, Helios, IFNγ, TNFα, CTLA-4, and Ki67 overnight at 4°C in permeabilization buffer. A complete list of antibodies can be found in Supplemental Table 1. Flow cytometry was performed on LSRII or Fortessa cytometers (BD Biosciences) and analyzed using FlowJo software v. 10.8 (BD Biosciences).

Spanier J.A., Fung V., Wardell C.M., Alkhatib M.H., Chen Y., Swanson L.A., Dwyer A.J., Weno M.E., Silva N., Mitchell J.S., Orban P.C., Mojibian M., Verchere C.B., Fife B.T, & Levings M.K. (2023). Insulin B peptide-MHC class II-specific chimeric antigen receptor-Tregs prevent autoimmune diabetes. bioRxiv.

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Multiparametric Flow Cytometry for T Cell Analysis

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Antibodies against the following proteins were purchased from BD Biosciences: CD69 (H1.2F3), CD122 (9TM-beta1), BCL-6 (K112-91), and CD127 (M1/69). Antibodies against the following proteins were purchased from eBioscience: CD44 (IM7), EOMES (Dan11mag), Ki-67 (SolA15), KLRG1 (2F1), and T-bet (eBio4B10). Antibodies against the following proteins were purchased from BioLegend: CD4 (RM4-5), CD27 (LG7F9), CD45 (30-F11), CD62L (MEL-14), PD-1 (29F.1A12), and Bcl2 (BCL/10C4). Phycoerythrin (PE)–labeled tetramer (MBL) staining for OVA-specific T cells was performed at a dilution of 1:50 before surface staining. Normal anti–TCF1/7 was purchased from Cell Signaling Technology. Fc Block (2.4G2) was purchased from BioXCell. Fixable viability dye eFluor780 was purchased from eBioscience; near-infrared fixable viability dye was purchased from Invitrogen. Flow cytometry experiments were performed on FACSCelesta (BD Biosciences) and analyzed using FlowJo software (v.10.3, Tree Star Inc.). Intracellular staining was performed with eBioscience Foxp3/Transcription Factor Staining Buffer Set (Invitrogen).

Rais R., Lemberg K.M., Tenora L., Arwood M.L., Pal A., Alt J., Wu Y., Lam J., Aguilar J.M., Zhao L., Peters D.E., Tallon C., Pandey R., Thomas A.G., Dash R.P., Seiwert T., Majer P., Leone R.D., Powell J.D, & Slusher B.S. (2022). Discovery of DRP-104, a tumor-targeted metabolic inhibitor prodrug. Science Advances, 8(46), eabq5925.

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Immunophenotyping of Iliac Lymph Nodes

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Harvested iliac lymph nodes were passed through a 70 μm strainer, washed with staining buffer (2% fetal bovine serum in 1× PBS; Serum Source International, Charlotte, NC, USA) and stained with antibodies for immunophenotyping (1:100 dilution; Table 1) in the presence of Fc block (2.4G2; BioXCell) for 30 min on ice, and analyzed on a BD LSR Fortessa X-20 (Becton Dickinson, Franklin Lakes, NJ, USA). Acquired data were analyzed using FlowJo software (Version 10, FlowJo LLC, Ashland, OR, USA). Dead cells were excluded using Ghost Dye-BV510 (Tonbo Biosciences, San Diego, CA, USA).

Boo B., Kamath R., Arriaga-Gomez E., Landry J., Emanuel E., Joo S., Saldías Montivero M., Martinov T., Fife B.T, & Chatterjea D. (2019). Tetrahydrocannabinol Reduces Hapten-Driven Mast Cell Accumulation and Persistent Tactile Sensitivity in Mouse Model of Allergen-Provoked Localized Vulvodynia. International Journal of Molecular Sciences, 20(9), 2163.

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