Helios 22f6

Manufactured by BioLegend

The Helios (22F6;) is a specialized lab equipment used for flow cytometry analysis. It serves as a core instrument for high-dimensional single-cell analysis. The Helios (22F6;) is designed to provide researchers with precise and reliable data on cellular phenotypes and functions.

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Close spelling variants of this product

Anti-Helios (22F6 (3 citations) Helios (clone 22F6, (3 citations) Helios-FITC (clone 22F6) (2 citations) Helios-Pacific Blue (22F6, (2 citations) HELIOS-FITC (22F6) (2 citations) Anti-mouse Helios (22F6, (2 citations)

8 protocols using helios 22f6

Comprehensive Antibody Panel for T-Cell Analysis

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The following antibodies were used from eBioscience (San Diego, CA) unless otherwise specified: Helios (22F6; Biolegend, San Diego, CA), CD90.1 (H1S51), TCRβ (H57-597; BD Biosciences, San Jose, CA), CD69 (H1.2F3), Vβ5 (MR9-4; BD Biosciences), Foxp3 (FJK16S), CD45RB (C363.16A), CD45.1 (A20), Vα2 (B20.1), CD3ε (eBio500A2), CD152 (UC10-4B9), folate receptor 4 (eBio12A5), CD73 (eBioTY/11.8), Vα2 (B20.1; BD Biosciences), CD25 (PC61.5), and CD4 (RM4-5; BD Biosciences).

Geem D., Ngo V., Harusato A., Chassaing B., Gewirtz A.T., Newberry R.D, & Denning T.L. (2016). Contribution of Mesenteric Lymph Nodes and GALT to the Intestinal Foxp3+ Regulatory T-Cell Compartment. Cellular and Molecular Gastroenterology and Hepatology, 2(3), 274-280.e3.

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T Cell Activation and Phenotyping

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Anti-CD3 (145–2C11), anti-CD28 (37.51), and anti-mouse CD16/CD32 (2.4G2) were purchased from BioXCell. Flow cytometry antibodies against CD4 (RM4–5), CD69 (H1.2F3), Nur77 (12.14), CD25 (PC61.5), and PD-1 (J43) were from eBioscience, against CD8α (53–6.7) and TCRβ (H57) from BD Pharmingen, and against Helios (22F6) from BioLegend. Live/dead fixable blue dead cell stain was from Invitrogen. Immunoblotting, antibodies against Nur77 (12.14), Helios (D8W4X), and β-Actin (AC-15) were from eBioscience, Cell Signaling, and Sigma, respectively. PE-conjugated α-galactosylceramide-loaded mouse CD1d tetramer was obtained from the NIH tetramer core facility.

Mittelstadt P.R., Taves M.D, & Ashwell J.D. (2019). Glucocorticoids oppose thymocyte negative selection by inhibiting Helios and Nur77. Journal of immunology (Baltimore, Md. : 1950), 203(8), 2163-2170.

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Cytokine and Transcription Factor Analysis

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Cytokines, transcriptional factors and surface markers were evaluated by flow cytometry with a FACSCanto II (BD Biosciences). To detect intracellular expression of IL-17A, IFN-γ in CD4⁺ T cells, lymph nodes or CNS (purified with Percoll) were first treated with 750 ng ml⁻¹ ionomycin (Sigma), 50 ng ml⁻¹ phorbol 12-myristate 13-acetate (PMA) (Sigma) and GolgiPlug (BD Biosciences) for 4–6 h at 37 °C. Cells were fixed and permeabilized with the Foxp3 Staining Buffer Set (eBioscience) or BD Cytofix/Cytoperm (BD Biosciences) and were stained with fluorescent antibodies. After washing, stained cells were assayed with a BD Biosciences FACSCanto II flow cytometer and data were analysed with FlowJo software. For flow cytometry, monoclonal antibodies against CD4 (clone GK1.5), CD8 (clone 53-6.7), CD62L (clone MEL-14), CD44 (clone IM7), CD25 (clone PC61.5), IL-17A (clone eBio17B7), IFN-γ (clone XMG1.2), Helios (22F6, Biolegend) and FoxP3 (clone FJK-16s) were from eBioscience and CD3 (clone 145-2C11) was from Biolegend.

Zhang L., Ke F., Liu Z., Bai J., Liu J., Yan S., Xu Z., Lou F., Wang H., Zhu H., Sun Y., Cai W., Gao Y., Li Q., Yu X.Z., Qian Y., Hua Z., Deng J., Li Q.J, & Wang H. (2015). MicroRNA-31 negatively regulates peripherally derived regulatory T-cell generation by repressing retinoic acid-inducible protein 3. Nature Communications, 6, 7639.

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Multiparametric Analysis of T Cell Subsets

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We stained human PBMCs with monoclonal antibodies to CD3 (UCHT1; BD Biosciences), CD4 (SK3; BD Biosciences), CD8 (SK1; BD Biosciences), CD25 (M-A251; BD Biosciences), CD45RA (ALB11; Beckman Coulter), CCR7 (MAB197; R&D Systems), Helios (22F6; BioLegend), Foxp3 (236A/E7; eBioscience), IL4 (MP4-25D2; BD Biosciences), IL-17 (eBio64DEC17; eBioscience), IFN-γ (B27; BD Biosciences) and a Fixable Viability Dye eFluor780 (eBioscience). The cytokine-producing capacity of T cell subsets was assessed after PBMCs were stimulated (at a density of 5 × 10⁵ cells per 100 μL) for 5 h with 50 ng/ml PMA (phorbol 12-myristate 13-acetate; Sigma-Aldrich) and 2 mg/ml of ionomycin (Sigma-Aldrich) in the presence of 10 mg/ml of brefeldin A (Sigma-Aldrich). Cells were fixed and made permeable with the Foxp3/Transcription Factor Staining Buffer Set according to the manufacturer's instructions (eBioscience). For flow cytometric analysis of human intestine, IEL and LPL were stained with monoclonal antibodies to CD3 (UCHT1; BD Biosciences), CD4 (SK3; BD Biosciences), CD8 (SK1; BD Biosciences), IL-7Rα (HIL-7R-M21; BD Pharmingen), and c-Kit (104D2; BD Biosciences).

Frascoli M., Jeanty C., Fleck S., Moradi P.W., Keating S., Mattis A.N., Tang Q, & MacKenzie T.C. (2016). Heightened immune activation in fetuses with gastroschisis may be blocked by targeting IL-5. Journal of immunology (Baltimore, Md. : 1950), 196(12), 4957-4966.

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Multiparametric Flow Cytometry for Immune Cell Profiling

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Samples were analyzed using Aurora (Cytek) and LSR Fortessa (Becton Dickinson) flow cytometers and the resulting data were analyzed using FlowJo software (Tree Star, Inc). The following monoclonal antibodies were used from eBioscience, CD3ε (145-2C11); CD4 (GK1.5); CD8a (53-6.7); CD45 (30-F11); Foxp3 (FJK-16s). Helios (22F6) and Ki67 (16A8) were from Biolegend. GITR was from BD. Nrp1 was purchased from R&D Systems.

Centa M., Weinstein E.G., Clemente J.C., Faith J.J., Fiel M.I., Lyallpuri R., Herbin O, & Alexandropoulos K. (2022). Impaired Central Tolerance Induces Changes in the Gut Microbiota that Exacerbate Autoimmune Hepatitis. Journal of autoimmunity, 128, 102808.

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T Cell Phenotyping by Flow Cytometry

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For surface staining, fluorophore-conjugated monoclonal Abs (mAbs) specific for CD3ε(17A2), TCRβ(H57–597), CD4(GK1.5 or RM4–5), PD-1 (RMP1–30), GITR (DTA-1), and CTLA-4 (UC10–4B9) were obtained from BioLegend. The mAb recognizing CXCR5 (2G8) was from BD Pharmingen. The mAb recognizing TCR Vβ6(RR4–7) was from eBioscience. For intranuclear staining, buffers from a Foxp3 Staining Buffer Set (eBioscience) were used to stain with Abs recognizing Foxp3 (FJK-16s, eBioscience), Helios (22F6, Biolegend), and/or Nur77 (12.14, eBioscience). Cells were run on an LSRII (BD Biosciences), and analyses were performed with FlowJo (TreeStar) software.

Confer D.L., Vercellotti G.M., Kotasek D., Goodman J.L., Ochoa A, & Jacob H.S. (1990). Herpes simplex virus-infected cells disarm killer lymphocytes.. Proceedings of the National Academy of Sciences of the United States of America, 87(9), 3609-3613.

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Comprehensive T cell phenotyping and analysis

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The following anti-mouse fluorochrome-conjugated mAbs and their respective isotype controls were used: CD3 (145-2C11), CD4 (clone RM4-5), CD8 (53-6.7), CD44 (IM7), CD49d (DX5), CD62L (MEL-14), B220 (RA3-6B2), IL-2 (JES6-5H4), IFN-γ (XMG1.2), IL-10 (JES5-16E3), IL-17 (TC11-18H10), MHC class II (I-A/I-E), PD-1 (CD279), all purchased from BD Biosciences; Helios (22F6) from Biolegend; and CD25 (PC61) and Foxp3 (FJK-16s) from eBioscience. For the detection of cytokines, cells from spleen and LN were resuspended at approximately 4 × 10⁶ cells/ml in RPMI-10% FCS, restimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 0.5 µg/ml ionomycin for 5 h, in the presence of 10 µg/ml Brefeldin A (Sigma). Cells were then harvested, surface stained, fixed for 10 min in BD FACS Lysing Solution (BD Biosciences), and washed and permeabilized with PermBuffer (eBioscience) before intracellular staining. Intracellular Foxp3 staining was performed using a Foxp3 staining kit (eBioscience). The staining of antigen-specific CD4⁺ T cells was carried out using the MHC class II I-Ab HY (116-130) NAGFNSNRANSSRSS tetramer (TCMetrix, Epalinges, Switzerland). Flow cytometry acquisition was done on FACS-Calibur™ using CellQuest™ (BectonDickinson) and data analyzed using FlowJo 9.5.3 software (Treestar).

Govender L., Wyss J.C., Kumar R., Pascual M, & Golshayan D. (2017). IL-2-Mediated In Vivo Expansion of Regulatory T Cells Combined with CD154–CD40 Co-Stimulation Blockade but Not CTLA-4 Ig Prolongs Allograft Survival in Naive and Sensitized Mice. Frontiers in Immunology, 8, 421.

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Transcription Factor and Cytokine Profiling

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For transcription factor staining, cells were treated as above then fixed and permeabilized with the eBioscience™ Intracellular Fixation and Permeabilization Buffer Set (Invitrogen) and incubated with fluorochrome-conjugated antibodies against FOXP3 (150D/E4; Thermo Fisher), active-Caspase-3 (C92-605; BD Pharmagen), Ki67 (16A8; BioLegend), Helios (22F6; BioLegend). For intra-cellular cytokine staining, a single cell suspension of cells was stimulated (Janssen et al., 2017 ), stained, fixed and permeabilized as above then incubated with fluorochrome-conjugated antibodies against FOXP3, T-bet (4B10; BioLegend), IFNγ (XMG1.2; Thermo Fisher), IL-10 (JES5-16E3; BioLegend). Cell counts of skin samples were determined by addition of Precision Count Beads™ (BioLegend) to samples immediately prior to flow cytometry. A BD LSRFortessa cell analyzer with FACSDiva software (BD Biosciences, San Jose, CA) was used to collect all data which was analyzed using FlowJo v10 (Tree Star Inc., San Jose, CA).

Wilkie H., Janssen E., Leyva-Castillo J.M, & Geha R.S. (2020). DOCK8 expression in Treg cells maintains their stability and limits contact hypersensitivity. The Journal of investigative dermatology, 141(6), 1503-1511.e3.

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