Two-color flow cytometric analysis of Helios expression Left Panel: Human peripheral blood mononuclear cells were fixed and permeabilized with the BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574/562725). The cells were then stained with PE Mouse Anti-Human CD4 antibody (Cat No. 555347/561843/561844) and either Alexa Fluor® 647 Hamster IgG2, κ Isotype Control (Cat No. 564073, Left Plot) or Alexa Fluor® 647 Armenian Hamster Anti-Helios antibody (Cat. No. 563951; Right Plot). Right Panel: Mouse splenic leucocytes were similarly fixed and permeabilized with the BD Transcription Factor Buffer Set. The cells were then stained with FITC Rat Anti-Mouse CD4 antibody (Cat No. 553729/557307/561828) and either Alexa Fluor® 647 Hamster IgG2, κ Isotype Control (Left Plot) or Alexa Fluor® 647 Armenian Hamster Anti-Helios antibody (Right Plot). The two-color flow cytometric dot plots show the correlated expression patterns of Helios (or Ig Isotype control staining) versus CD4 for gated events with the forward and side light-scatter characteristics of intact human peripheral blood lymphocytes or splenic leucocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Two-color flow cytometric analysis of Helios expression Left Panel: Human peripheral blood mononuclear cells were fixed and permeabilized with the BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574/562725). The cells were then stained with PE Mouse Anti-Human CD4 antibody (Cat No. 555347/561843/561844) and either Alexa Fluor® 647 Hamster IgG2, κ Isotype Control (Cat No. 564073, Left Plot) or Alexa Fluor® 647 Armenian Hamster Anti-Helios antibody (Cat. No. 563951; Right Plot). Right Panel: Mouse splenic leucocytes were similarly fixed and permeabilized with the BD Transcription Factor Buffer Set. The cells were then stained with FITC Rat Anti-Mouse CD4 antibody (Cat No. 553729/557307/561828) and either Alexa Fluor® 647 Hamster IgG2, κ Isotype Control (Left Plot) or Alexa Fluor® 647 Armenian Hamster Anti-Helios antibody (Right Plot). The two-color flow cytometric dot plots show the correlated expression patterns of Helios (or Ig Isotype control staining) versus CD4 for gated events with the forward and side light-scatter characteristics of intact human peripheral blood lymphocytes or splenic leucocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Product Details
BD Pharmingen™
IKAROS family zinc finger protein 2; ANF1A2; HELIOS; ZNF1A2; ZNFN1A2
Human (QC Testing), Mouse (Tested in Development)
Armenian Hamster IgG
Mouse Helios Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_2738506
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO
Preparation And Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Although hamster immunoglobulin isotypes have not been well defined, BD Biosciences Pharmingen has grouped Armenian and Syrian hamster IgG monoclonal antibodies according to their reactivity with a panel of mouse anti-hamster IgG mAbs. A table of the hamster IgG groups, Reactivity of Mouse Anti-Hamster Ig mAbs, may be viewed at http://www.bdbiosciences.com/documents/hamster_chart_11x17.pdf.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Transcription Factor Buffer Set RUO
Size
100 Tests
Cat No.
562574
Alexa Fluor® 647 Hamster IgG2, κ Isotype Control RUO
Size
50 µg
Cat No.
564073
Transcription Factor Buffer Set RUO
Size
25 Tests
Cat No.
562725
PE Mouse Anti-Human CD4 RUO
Size
100 Tests
Cat No.
555347
563951 Rev. 2
Antibody Details
22F6
The 22F6 monoclonal antibody specifically binds to mouse and human Helios. Helios is a member of the Ikaros family of zinc-finger transcription factors, which play important roles in hematopoietic cell development and tumor suppression. Helios expression is restricted to the earliest stages of embryonic hematopoiesis, a variety of epithelial tissues and is notably increased in thymic-derived regulatory CD4+Foxp3+ T (Treg) cells. Its high expression levels in Treg cells are independent from Foxp3 and are believed to contribute, along with other transcription factors, to the phenotypic stability of natural regulatory T cells. Accordingly, it has been demonstrated that Helios directly stimulates Foxp3 transcription while it inhibits ll2 gene expression, contributing for the maintenance of cellular anergy. Helios is also differentially expressed during negative and positive selection in the thymus, marking CD4+ autoreactive cells for deletion. Helios may possibly play roles in T cell activation, since it is upregulated in Th2 and Tfh cells. Despite these roles in T cell development and function, Helios genetic ablation in mice revealed no significant abnormalities in Treg or other T cell subsets. This finding suggests that other Ikaros family members may play redundant roles.
563951 Rev. 2
Format Details
Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
563951 Rev.2
Citations & References
Development References (9)
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Baine I, Basu S, Ames R, Sellers RS, Macian F. Helios induces epigenetic silencing of IL2 gene expression in regulatory T cells. J Immunol. 2013; 190(3):1008-1016. (Biology). View Reference
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Cai Q, Dierich A, Oulad-Abdelghani M, Chan S, Kastner P. Helios deficiency has minimal impact on T cell development and function. J Immunol. 183(4)(Biology). View Reference
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Daley SR, Hu DY, Goodnow CC. Helios marks strongly autoreactive CD4+ T cells in two major waves of thymic deletion distinguished by induction of PD-1 or NF-kappaB. J Exp Med. 2013; 210(2):269-285. (Biology). View Reference
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Fu W, Ergun A, Lu T, et al. A multiply redundant genetic switch 'locks in' the transcriptional signature of regulatory T cells. Nat Immunol. 2012; 13(10):972-980. (Biology). View Reference
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Georgopoulos K. Haematopoietic cell-fate decisions, chromatin regulation and ikaros. Nat Rev Immunol. 2002; 2(3):162-174. (Biology). View Reference
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Kelley CM, Ikeda T, Koipally J, et al. Helios, a novel dimerization partner of Ikaros expressed in the earliest hematopoietic progenitors. Curr Biol. 1998; 8(9):508-515. (Biology). View Reference
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Serre K, Benezech C, Desanti G, et al. Helios is associated with CD4 T cells differentiating to T helper 2 and follicular helper T cells in vivo independently of Foxp3 expression. PLoS ONE. 2011; 6(6):e20731. (Biology). View Reference
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Sugimoto N, Oida T, Hirota K, et al. Foxp3-dependent and -independent molecules specific for CD25+CD4+ natural regulatory T cells revealed by DNA microarray analysis. Int Immunol. 18(8)(Biology). View Reference
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Thornton AM, Korty PE, Tran DQ, et al. Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J Immunol. 2010; 184(7):3433-3441. (Immunogen: ELISA, Flow cytometry, Western blot). View Reference
563951 Rev. 2
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
