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BD Horizon™ BV421 Mouse Anti-Oct3/4
Clone 40/Oct-3 (RUO)
Flow cytometric analysis and immunofluorescent staining of Oct3/4 expression in human embryonic stem (ES) cells.
LEFT: H9 human ES cells (WiCell, Madison, WI) grown in mTESR™1 media (StemCell Technologies) were harvested using Accutase™ Cell Detachment Solution (Cat. No. 561527), fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050). Cells were stained with either BD Horizon™ BV421 Mouse anti-Oct3/4 (Cat. No. 565644, solid line) or BD Horizon™ BV421 Mouse IgG1, κ Isotype Control (Cat. No. 562438, dashed line) at matched concentrations. Histograms were derived from gated events based on light scattering characteristics of human ES cells. Flow cytometry was performed on a BD LSRFortessa™ Flow Cytometry System.
RIGHT: H9 human ES cells (WiCell, Madison, WI) were cultured in a 96-well imaging plate, fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050), and blocked with 5% goat serum, 1% BSA, and 0.5% Triton™ X-100 diluted in 1× PBS. Cells were stained with BD Horizon™ BV421 Mouse Anti-Human Oct3/4 antibody (Cat. No. 565644, pseudo-colored red) and counterstained with BD Pharmingen™ DRAQ5™ (Cat. No. 564903, pseudo-colored blue). Images were captured on a standard epifluorescence microscope. Original magnification, 20×.
Flow cytometric analysis and immunofluorescent staining of Oct3/4 expression in human embryonic stem (ES) cells.
LEFT: H9 human ES cells (WiCell, Madison, WI) grown in mTESR™1 media (StemCell Technologies) were harvested using Accutase™ Cell Detachment Solution (Cat. No. 561527), fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050). Cells were stained with either BD Horizon™ BV421 Mouse anti-Oct3/4 (Cat. No. 565644, solid line) or BD Horizon™ BV421 Mouse IgG1, κ Isotype Control (Cat. No. 562438, dashed line) at matched concentrations. Histograms were derived from gated events based on light scattering characteristics of human ES cells. Flow cytometry was performed on a BD LSRFortessa™ Flow Cytometry System.
RIGHT: H9 human ES cells (WiCell, Madison, WI) were cultured in a 96-well imaging plate, fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050), and blocked with 5% goat serum, 1% BSA, and 0.5% Triton™ X-100 diluted in 1× PBS. Cells were stained with BD Horizon™ BV421 Mouse Anti-Human Oct3/4 antibody (Cat. No. 565644, pseudo-colored red) and counterstained with BD Pharmingen™ DRAQ5™ (Cat. No. 564903, pseudo-colored blue). Images were captured on a standard epifluorescence microscope. Original magnification, 20×.
Flow cytometric analysis and immunofluorescent staining of Oct3/4 expression in human embryonic stem (ES) cells.
LEFT: H9 human ES cells (WiCell, Madison, WI) grown in mTESR™1 media (StemCell Technologies) were harvested using Accutase™ Cell Detachment Solution (Cat. No. 561527), fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050). Cells were stained with either BD Horizon™ BV421 Mouse anti-Oct3/4 (Cat. No. 565644, solid line) or BD Horizon™ BV421 Mouse IgG1, κ Isotype Control (Cat. No. 562438, dashed line) at matched concentrations. Histograms were derived from gated events based on light scattering characteristics of human ES cells. Flow cytometry was performed on a BD LSRFortessa™ Flow Cytometry System.
RIGHT: H9 human ES cells (WiCell, Madison, WI) were cultured in a 96-well imaging plate, fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050), and blocked with 5% goat serum, 1% BSA, and 0.5% Triton™ X-100 diluted in 1× PBS. Cells were stained with BD Horizon™ BV421 Mouse Anti-Human Oct3/4 antibody (Cat. No. 565644, pseudo-colored red) and counterstained with BD Pharmingen™ DRAQ5™ (Cat. No. 564903, pseudo-colored blue). Images were captured on a standard epifluorescence microscope. Original magnification, 20×.
Flow cytometric analysis and immunofluorescent staining of Oct3/4 expression in human embryonic stem (ES) cells.
LEFT: H9 human ES cells (WiCell, Madison, WI) grown in mTESR™1 media (StemCell Technologies) were harvested using Accutase™ Cell Detachment Solution (Cat. No. 561527), fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050). Cells were stained with either BD Horizon™ BV421 Mouse anti-Oct3/4 (Cat. No. 565644, solid line) or BD Horizon™ BV421 Mouse IgG1, κ Isotype Control (Cat. No. 562438, dashed line) at matched concentrations. Histograms were derived from gated events based on light scattering characteristics of human ES cells. Flow cytometry was performed on a BD LSRFortessa™ Flow Cytometry System.
RIGHT: H9 human ES cells (WiCell, Madison, WI) were cultured in a 96-well imaging plate, fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050), and blocked with 5% goat serum, 1% BSA, and 0.5% Triton™ X-100 diluted in 1× PBS. Cells were stained with BD Horizon™ BV421 Mouse Anti-Human Oct3/4 antibody (Cat. No. 565644, pseudo-colored red) and counterstained with BD Pharmingen™ DRAQ5™ (Cat. No. 564903, pseudo-colored blue). Images were captured on a standard epifluorescence microscope. Original magnification, 20×.
Product Details
BD Horizon™
Oct3, OTF3, Oct4, OTF4, POU5F1
Human (QC Testing), Mouse (Tested in Development)
Mouse IgG1, κ
Mouse Oct3 aa. 252-372 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested), Immunofluorescence (Tested During Development)
0.2 mg/ml
AB_2739320
Aqueous buffered solution containing BSA and ≤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 with BD Horizon BV421 under optimum conditions, and unconjugated antibody and free BD Horizon BV421 were removed.
Recommended Assay Procedures
For optimal and reproducible results, BD Horizon Brilliant™ Stain Buffer should be used anytime BD Horizon Brilliant™ dyes are used in a multicolor flow cytometry panel. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. When BD Horizon Brilliant Stain Buffer is used in in the multicolor panel, it should also be used in the corresponding compensation controls for all dyes to achieve the most accurate compensation. For the most accurate compensation, compensation controls created with either cells or beads should be exposed to BD Horizon Brilliant Stain Buffer for the same length of time as the corresponding multicolor panel. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
- Accutase is a registered trademark of Innovative Cell Technologies, Inc.
- DRAQ5™ is a registered trademark of BioStatus Ltd.
- mTESR™1 is a trademark of StemCell Technologies.
- Triton is a trademark of the Dow Chemical Company.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- An isotype control should be used at the same concentration as the antibody of interest.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
Companion Products
Stain Buffer (FBS) RUO
Size
500 ML
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 ML
Cat No.
554657
Brilliant Stain Buffer RUO
Size
100 Tests
Cat No.
563794
Fixation Buffer RUO
Size
100 ML
Cat No.
554655
Perm Buffer III RUO
Size
125 ML
Cat No.
558050
BV421 Mouse IgG1, k Isotype Control RUO
Size
50 µg
Cat No.
562438
565644 Rev. 2
Antibody Details
40/Oct-3
Development of a multicellular organism from a single fertilized cell is regulated by the coordinated activity of DNA transcription factors. Oct3/4, a member of the POU family of transcription factors, functions in pluripotent cells of early embryonic stem cell (ES) lines and embryonal carcinomas (EC). Other members of the POU family include Oct1, Oct2, Pit-1, and unc-86. The POU domain, a 150-amino acid region that determines binding specificity, is conserved among these proteins and consists of 3 subdomains: POU-specific A and B subdomains and a homeobox-like subdomain. Oct3/4 is expressed in undifferentiated cells, but is lost as cells are induced to differentiate. Oct3/4 is not expressed in adult tissues. The interaction of Oct3/4 with SOX2, another embryonic transcription factor, produces an active complex that regulates expression of genes such as Nanog, UTF1, and FGF4. Although Oct3/4 is specifically phosphorylated on serine residues, this modification is not required for DNA binding, but may affect its transactivation potential. Thus, Oct3/4 is a transcription factor that plays an important role in determining early steps of embryogenesis and differentiation.
565644 Rev. 2
Format Details
BV421
The BD Horizon Brilliant Violet™ 421 (BV421) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This polymer-technology based dye has an excitation maximum (Ex Max) of 407-nm and an emission maximum (Em Max) at 423-nm. Driven by BD innovation, BV421 is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 420-nm (e.g., a 431/28-nm or 450/50-nm bandpass filter). BV421 is an ideal alternative for V450 as it is approximately ten times brighter with less spillover into the BV510/V500 detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
BV421
407 nm
423 nm
565644 Rev.2
Citations & References
View product citations for antibody "565644" on CiteAb
Development References (5)
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Nishimoto M, Fukushima A, Okuda A, Muramatsu M. The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2. Mol Cell Biol. 1999; 19(8):5453-5465. (Biology). View Reference
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Okamoto K, Okazawa H, Okuda A, Sakai M, Muramatsu M, Hamada H. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell. 1990; 60(3):461-472. (Biology). View Reference
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Pan G, Thomson JA. Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res. 2007; 17:42-49. (Biology). View Reference
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Rosfjord E, Scholtz B, Lewis R, Rizzino A. Phosphorylation and DNA binding of the octamer binding transcription factor Oct-3. Biochem Biophys Res Commun. 1995; 212(3):847-853. (Biology). View Reference
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Vigano MA, Staudt LM. Transcriptional activation by Oct-3: evidence for a specific role of the POU-specific domain in mediating functional interaction with Oct-1. Nucleic Acids Res. 1996; 24(11):2112-2118. (Biology). View Reference
565644 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.