Fc block 2.4g2, by BD - Product details

1

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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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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3

Intestinal Epithelial Cell FACS Isolation

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FACS analysis and cell sorting were performed as described previously [48] (link). In brief, the small intestine was cut longitudinally and washed in cold PBS. Then, a 2-cm piece of intestine was cut and incubated in 2% (vol/vol) fetal calf serum in RPMI 1640 (Wako) containing 0.5 mM EDTA (Nacalai Tesque) for 15 minutes at 37°C. Cells were filtered with a 70-μm cell strainer (Becton Dickinson) and blocked with Fc Block (2.4G2; Becton Dickinson) for 5 minutes at room temperature, followed by staining antibodies and reagents for 30 minutes at 4°C. Dead cells were excluded by propidium iodide (PI) staining. Epithelial cell fraction was defined by PI⁻CD45⁻TER119⁻EpCAM⁺ gating and biotinylated HKH-189 was used for Cld4 staining. Samples were analyzed by FACSCanto (Becton Dickinson) or sorted by FACSAria (Becton Dickinson).

Nagatake T., Fujita H., Minato N, & Hamazaki Y. (2014). Enteroendocrine Cells Are Specifically Marked by Cell Surface Expression of Claudin-4 in Mouse Small Intestine. PLoS ONE, 9(3), e90638.

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4

Isolation and Culture of Immune Cells

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Blood was collected by cheek bleeding or intracardial puncture. Spleens were smashed through a 70 μm nylon mesh filter (Becton Dickinson). Primary BM cells were harvested by flushing femurs' content using IMDM (Gibco). Red blood cells were lysed in an ammonium chloride buffer, and peritoneal lavages were harvested, as described elsewhere (29 (link)). For skin cell preparations, ears were split into the dorsal and ventral layers, minced and digested 30 min at 37C in a shaking incubator (150 rpm) in IMDM 5% FCS (Gibco) containing 2 mg/mL of collagenase IV and 100 μg/mL DNAse I (both Sigma). Digestion was stopped by adding 5 mM EDTA and a single cell suspension obtained by smashing the remaining tissue through a 70 μm nylon mesh filter (BD). Cells were cultured ex vivo in DMEM 20% FCS + Non-essential amino acid + Sodium pyruvate (Gibco) at 37C +5% CO₂. Peritoneal lavages incubation with DT was done in the presence of 10 ng/mL of Stem cell factor (SCF) (Peprotech). Whole splenocytes were stimulated with IL3 (Peprotech) or sterile-filtered FcBlock (2.4G2, Becton Dickinson, 10 μg/mL), MAR1 (0.5 μg/mL) or Ba13-APC (Biolegend, 1 μg/mL), Phorbol–myristate acetate (PMA, 100 ng/mL), ionomycin (100 ng/mL) or both (Thermofischer) for the indicated times in culture medium.

Pellefigues C., Mehta P., Prout M.S., Naidoo K., Yumnam B., Chandler J., Chappell S., Filbey K., Camberis M, & Le Gros G. (2019). The Basoph8 Mice Enable an Unbiased Detection and a Conditional Depletion of Basophils. Frontiers in Immunology, 10, 2143.

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5

Comprehensive Immune Cell Profiling

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Cells were stained with Ghost Violet™ 510 Viability Dye (TONBO Biosciences) for dead cell exclusion, blocked with Fc block (2.4G2, BD Biosciences) and stained with the following anti-mouse antibodies from Biolegend: rat anti-CD4-BV785 (GK1.5), rat anti-CD44-PE-Cy7 (IM7), hamster anti-TCRb-APC (H57-597), rat anti-CD8-PerCP-Cy5.5 (53-6.7), rat anti-CD45-APC-Cy7 (30-F11), rat anti-CD25-FITC (3C7), rat anti-CD5-A700 (53-7.3), rat anti-CD127-BV421 (A7R34) and hamster anti-CD69-PE (H1.2F3) (for thymus samples only) or rat anti-CD62L-PE (MEL-14) (for spleen and lymph node samples only). All antibodies were diluted at 1/200, except for anti-CD4-BV785 (1/400) and anti-CD44-PE-Cy7 (1/700). AccuCheck counting beads (Thermo Fisher Scientific) were added to each sample in order to calculate absolute cell numbers. Samples were analysed on a Fortessa (BD Biosciences) instrument.

Evnouchidou I., Chappert P., Benadda S., Zucchetti A., Weimershaus M., Bens M., Caillens V., Koumantou D., Lotersztajn S., van Endert P., Davoust J., Guermonprez P., Hivroz C., Gross D.A, & Saveanu L. (2020). IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses. Nature Communications, 11, 2779.

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6

Multiparameter Flow Cytometry of Immune Cells

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The following fluorophore conjugated antibodies were purchased from eBioscience: T- bet (4B10), Foxp3 (FJK-16s), MHCII (M5/114.15.2), CD11b (M1/70), IL-12p40 (C17.8), CD62L (MEL-14), CD25 (PC61.5), Rat IgG1, Rat IgG2a, Rat IgG2b and Mouse IgG1. The following antibodies were purchased from BD Biosciences: IFNγ (XMG1.2), CD103 (M290), CD45 (30-F11), CD152 (UC10-4F10-11), and Fc Block^™ (2.4G2). The following antibodies were purchased from Biolegend: CD4 (GK1.5), TCRb (H57-597), CD45.1 (A20), CD11c (N418), CD8a (53-6.7), CD86 (GL-1), IL-10 (JES5-16E3), and CD44 (IM7). Aqua LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit was purchased from Life Technologies. Cells were permeabilized with the Foxp3 fixation/permeabilization kit for transcription factor (eBioscience) or Cytofix/Cytoperm (BD Biosciences) for cytokine staining. Flow cytometry was performed with a 9-color BD FACSCanto (BD Biosciences) and data were analyzed using FlowJo software (Treestar). Sorting experiments were performed with an Aria Fusion (BD Biosciences).

Bouziat R., Biering S.B., Kouame E., Sangani K.A., Kang S., Ernest J.D., Varma M., Brown J.J., Urbanek K., Dermody T.S., Ng A., Hinterleitner R., Hwang S, & Jabri B. (2018). Murine Norovirus infection induces TH1 inflammatory responses to dietary antigens. Cell host & microbe, 24(5), 677-688.e5.

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7

Multiparameter Flow Cytometric Analysis

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Fluorescence dye labeled antibodies (Abs) specific for CD3 (145-2C11), CD4 (L3T4), CD25 (PC61.5), CD45 (30F11), CD45RB (C363.16A), CD11c (N418), TCRβ (H57-597), MHC-II (M5/114.15.2), Helios (22F6), FoxP3 (NRRF-30), IL-9 (RM9A4), pSMAD2/3 (O72-670), pSTAT5 (SRBCZX), pSTAT6 (18/P-Stat6), PU.1 (9G7), and IRF4 (3E4) were purchased from Becton Dickinson (BD), eBioscience, Biolegend and Cell Signaling Technology. Fc block (2.4G2) was purchased from BD. Dead cells were identified using the fixable Aqua dead cell staining kit (Invitrogen). Intracellular staining for Helios, IRF4 and Foxp3 was performed using a Foxp3 staining buffer set (eBioscience). Intracellular staining of IL-9 was performed after restimulation of cells with phorbol-12-myristate 13-acetate (Sigma), ionomycin (Sigma) and brefeldin A (eBioscience) for 4 hours. Stimulated cells were fixed and permeabilized, and then stained with Abs specific for IL-9. Detection of pSMAD2/3 was performed according to the BD Phosflow protocol. For intracellular detection of pSTAT5, pSTAT6 and PU.1, cells were fixed by 1.6% paraformaldehyde and incubated for 10 min at room temperature. Cells were then permeabilized by ice-cold methanol and stored at −80 °C before staining. Multi-parameter analysis was performed on a Fortessa (BD) and analyzed with FlowJo software (Tree Star). Cell sorting was performed using a SH800Z cell sorter (SONY).

Harusato A., Abo H., Le Ngo V., Yi S.W., Mitsutake K., Osuka S., Kohlmeier J.E., Li J.D., Gewirtz A.T., Nusrat A, & Denning T.L. (2017). IL-36γ signaling controls the induced regulatory T cell – TH9 cell balance via NFκB activation and STAT transcription factors. Mucosal immunology, 10(6), 1455-1467.

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Multiparameter Flow Cytometric Analysis

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Fluorescence dye labeled antibodies (Abs) specific for CD3 (145-2C11), CD4 (L3T4), CD25 (PC61.5), CD45 (30F11), CD45RB (C363.16A), CD11c (N418), TCRβ (H57-597), MHC-II (M5/114.15.2), Helios (22F6), FoxP3 (NRRF-30), IL-9 (RM9A4), pSMAD2/3 (O72-670), pSTAT5 (SRBCZX), pSTAT6 (18/P-Stat6), PU.1 (9G7), and IRF4 (3E4) were purchased from Becton Dickinson (BD), eBioscience, Biolegend and Cell Signaling Technology. Fc block (2.4G2) was purchased from BD. Dead cells were identified using the fixable Aqua dead cell staining kit (Invitrogen). Intracellular staining for Helios, IRF4 and Foxp3 was performed using a Foxp3 staining buffer set (eBioscience). Intracellular staining of IL-9 was performed after restimulation of cells with phorbol-12-myristate 13-acetate (Sigma), ionomycin (Sigma) and brefeldin A (eBioscience) for 4 hours. Stimulated cells were fixed and permeabilized, and then stained with Abs specific for IL-9. Detection of pSMAD2/3 was performed according to the BD Phosflow protocol. For intracellular detection of pSTAT5, pSTAT6 and PU.1, cells were fixed by 1.6% paraformaldehyde and incubated for 10 min at room temperature. Cells were then permeabilized by ice-cold methanol and stored at −80 °C before staining. Multi-parameter analysis was performed on a Fortessa (BD) and analyzed with FlowJo software (Tree Star). Cell sorting was performed using a SH800Z cell sorter (SONY).

Harusato A., Abo H., Le Ngo V., Yi S.W., Mitsutake K., Osuka S., Kohlmeier J.E., Li J.D., Gewirtz A.T., Nusrat A, & Denning T.L. (2017). IL-36γ signaling controls the induced regulatory T cell – TH9 cell balance via NFκB activation and STAT transcription factors. Mucosal immunology, 10(6), 1455-1467.

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9

Multiparameter Flow Cytometry for Immune Cell Profiling

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Single-cell suspensions in FACs buffer were incubated in Fc block (2.4G2, BD Biosciences, Franklin Lakes, NJ, USA) for 15 min then samples were washed in FACs buffer and centrifuged at 400 × g for 4 min. Cells were then incubated in a solution containing fluorescently labelled antibodies for 30 min in the dark on ice. Following staining, flow cytometry was performed on a BD FACS Canto II (BD Biosciences) or a LSRFortessa™ (BD Biosciences). Analysis was performed on FlowJo software (BD Biosciences) using the gating strategy outlined in Additional file 2: Fig S2.
The following antibodies were used for the detection and phenotyping of immune cells: CD45-BV510 (30-F11; Biolegend, San Diego, CA, USA), CD3-APC (17A2; Biolegend), B220-APC-Cy7 (RA3-6B2; Biolegend), CD4-BV421 (RM4-5; Biolegend), CD8a-PerCP-Cy5.5 (53–6.7; Biolegend), CD62L-FITC (MEL-14; Biolegend), CD44-PE (IM7; Biolegend), CD11b-PE-Cy7 (M1/70; Biolegend), Ly6G-APC (1A8; Biolegend), Ly6C-PE (HK1.4; Biolegend), and CD49d-AF488 (R1-2; Biolegend).

Peck T., Davis C., Lenihan-Geels G., Griffiths M., Spijkers-Shaw S., Zubkova O.V, & La Flamme A.C. (2023). The novel HS-mimetic, Tet-29, regulates immune cell trafficking across barriers of the CNS during inflammation. Journal of Neuroinflammation, 20, 251.

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Multiparameter Flow Cytometry Analysis

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BALF or lung mononuclear cells were stained at 4°C in staining buffer (1X PBS, 2% FCS, 2mM EDTA), in the presence of Fc block (2.4G2; BD Biosciences) and analyzed by flow cytometry.
Cells were incubated with CD1d-PBS57-APC tetramers and/or the specific antibodies listed below. For intracellular staining, cells were further fixed with 4% PFA, washed, and permeabilized with 0.5% saponin (Sigma-Aldrich), and then incubated with the anti-cytokine antibodies. The cells were washed and fluorescence was detected using a LSRFortessa (Becton Dickinson). Data were analyzed using the FlowJo 10.4.1 software (Tree Star). Figure S1 represents the gate strategy used.
Antibodies from BD Biosciences: anti-CD3-FITC (145-2C11), anti-CD45-APC-Cy7 (30F11). Antibodies from BioLegend: anti-CD4-Brilliant Violet 605 (RM4-5), anti-CD69-FITC (H1.2F3), anti-CD8a-Brilliant Violet 785 (53-6.7), anti-TCR Vγ1/Cr4-PE (2.11) [Tonegawa 1986 nomenclature, (21 (link))]. Antibodies from eBioscience: anti-CD44-eFluor450 (IM7), anti-TCRb-AlexaFluor 700 (H57-598), anti-IL-13-PE-eFluor610 (eBio13A), anti-IL-17A-PerCP-Cy5.5 (eBio17B7), anti-IL-4-PE-Cy7 (BVD6-24G2), anti-TCRδ-eFluor450 and -PE-Cy7 (GL3), and Fixable Viability Dye eFluor 506.

Foray A.P., Dietrich C., Pecquet C., Machavoine F., Chatenoud L, & Leite-de-Moraes M. (2021). IL-4 and IL-17 Are Required for House Dust Mite-Driven Airway Hyperresponsiveness in Autoimmune Diabetes-Prone Non-Obese Diabetic Mice. Frontiers in Immunology, 11, 595003.

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Fc block 2.4g2

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41 protocols using fc block 2.4g2

Quantifying Human Cell Engraftment

Quantifying Human Cell Engraftment

Intestinal Epithelial Cell FACS Isolation

Isolation and Culture of Immune Cells

Comprehensive Immune Cell Profiling

Multiparameter Flow Cytometry of Immune Cells

Multiparameter Flow Cytometric Analysis

Multiparameter Flow Cytometric Analysis

Multiparameter Flow Cytometry for Immune Cell Profiling

Multiparameter Flow Cytometry Analysis

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