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BD Pharmingen™ Alexa Fluor® 647 Mouse Anti-Nestin
Clone 25/NESTIN (RUO)
LEFT Image: Analysis of Alexa Fluor® 647 Mouse Anti-Nestin on human Neural Stem cells (NSCs). H9-derived NSCs were isolated by sorting from Embryoid bodies and were grown for 2 passages post sort, fixed (BD Cytofix™ Fixation Buffer, Cat. No. 554655) for 20 minutes at room temperature, permeabilized with BD Phosflow™ Perm Buffer I (Cat. No.557885), and then stained with either Alexa Fluor® 647 Mouse anti-Nestin (Cat. No. 560393; solid line histogram) or Alexa Fluor® 647 Mouse IgG1, κ Isotype Control (Cat. No. 557732; dashed line histogram). Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
RIGHT Image: Analysis of Alexa Fluor® 647 Mouse Anti-Nestin on human neurons. H9-derived NSC were differentiated into neurons for 12 days. The cells were fixed (BD Cytofix™ Fixation Buffer) for 20 minutes at room temperature, permeabilized with BD Phosflow™ Perm Buffer I, and then stained with either Alex Fluor® 647 Mouse anti-Nestin (solid line histogram) or Alexa Fluor® 647 Mouse IgG1, k Isotype Control (dashed line histogram). The two peaks reveal differentiated neurons (low Nestin staining) and undifferentiated NSC (high Nestin staining). This antibody also works in BD Phosflow™ Perm Buffers II and III. Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
LEFT Image: Analysis of Alexa Fluor® 647 Mouse Anti-Nestin on human Neural Stem cells (NSCs). H9-derived NSCs were isolated by sorting from Embryoid bodies and were grown for 2 passages post sort, fixed (BD Cytofix™ Fixation Buffer, Cat. No. 554655) for 20 minutes at room temperature, permeabilized with BD Phosflow™ Perm Buffer I (Cat. No.557885), and then stained with either Alexa Fluor® 647 Mouse anti-Nestin (Cat. No. 560393; solid line histogram) or Alexa Fluor® 647 Mouse IgG1, κ Isotype Control (Cat. No. 557732; dashed line histogram). Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
RIGHT Image: Analysis of Alexa Fluor® 647 Mouse Anti-Nestin on human neurons. H9-derived NSC were differentiated into neurons for 12 days. The cells were fixed (BD Cytofix™ Fixation Buffer) for 20 minutes at room temperature, permeabilized with BD Phosflow™ Perm Buffer I, and then stained with either Alex Fluor® 647 Mouse anti-Nestin (solid line histogram) or Alexa Fluor® 647 Mouse IgG1, k Isotype Control (dashed line histogram). The two peaks reveal differentiated neurons (low Nestin staining) and undifferentiated NSC (high Nestin staining). This antibody also works in BD Phosflow™ Perm Buffers II and III. Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
LEFT Image: Analysis of Alexa Fluor® 647 Mouse Anti-Nestin on human Neural Stem cells (NSCs). H9-derived NSCs were isolated by sorting from Embryoid bodies and were grown for 2 passages post sort, fixed (BD Cytofix™ Fixation Buffer, Cat. No. 554655) for 20 minutes at room temperature, permeabilized with BD Phosflow™ Perm Buffer I (Cat. No.557885), and then stained with either Alexa Fluor® 647 Mouse anti-Nestin (Cat. No. 560393; solid line histogram) or Alexa Fluor® 647 Mouse IgG1, κ Isotype Control (Cat. No. 557732; dashed line histogram). Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
RIGHT Image: Analysis of Alexa Fluor® 647 Mouse Anti-Nestin on human neurons. H9-derived NSC were differentiated into neurons for 12 days. The cells were fixed (BD Cytofix™ Fixation Buffer) for 20 minutes at room temperature, permeabilized with BD Phosflow™ Perm Buffer I, and then stained with either Alex Fluor® 647 Mouse anti-Nestin (solid line histogram) or Alexa Fluor® 647 Mouse IgG1, k Isotype Control (dashed line histogram). The two peaks reveal differentiated neurons (low Nestin staining) and undifferentiated NSC (high Nestin staining). This antibody also works in BD Phosflow™ Perm Buffers II and III. Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
LEFT Image: Analysis of Alexa Fluor® 647 Mouse Anti-Nestin on human Neural Stem cells (NSCs). H9-derived NSCs were isolated by sorting from Embryoid bodies and were grown for 2 passages post sort, fixed (BD Cytofix™ Fixation Buffer, Cat. No. 554655) for 20 minutes at room temperature, permeabilized with BD Phosflow™ Perm Buffer I (Cat. No.557885), and then stained with either Alexa Fluor® 647 Mouse anti-Nestin (Cat. No. 560393; solid line histogram) or Alexa Fluor® 647 Mouse IgG1, κ Isotype Control (Cat. No. 557732; dashed line histogram). Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
RIGHT Image: Analysis of Alexa Fluor® 647 Mouse Anti-Nestin on human neurons. H9-derived NSC were differentiated into neurons for 12 days. The cells were fixed (BD Cytofix™ Fixation Buffer) for 20 minutes at room temperature, permeabilized with BD Phosflow™ Perm Buffer I, and then stained with either Alex Fluor® 647 Mouse anti-Nestin (solid line histogram) or Alexa Fluor® 647 Mouse IgG1, k Isotype Control (dashed line histogram). The two peaks reveal differentiated neurons (low Nestin staining) and undifferentiated NSC (high Nestin staining). This antibody also works in BD Phosflow™ Perm Buffers II and III. Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
LEFT Image: Analysis of Alexa Fluor® 647 Mouse Anti-Nestin on human Neural Stem cells (NSCs). H9-derived NSCs were isolated by sorting from Embryoid bodies and were grown for 2 passages post sort, fixed (BD Cytofix™ Fixation Buffer, Cat. No. 554655) for 20 minutes at room temperature, permeabilized with BD Phosflow™ Perm Buffer I (Cat. No.557885), and then stained with either Alexa Fluor® 647 Mouse anti-Nestin (Cat. No. 560393; solid line histogram) or Alexa Fluor® 647 Mouse IgG1, κ Isotype Control (Cat. No. 557732; dashed line histogram). Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
RIGHT Image: Analysis of Alexa Fluor® 647 Mouse Anti-Nestin on human neurons. H9-derived NSC were differentiated into neurons for 12 days. The cells were fixed (BD Cytofix™ Fixation Buffer) for 20 minutes at room temperature, permeabilized with BD Phosflow™ Perm Buffer I, and then stained with either Alex Fluor® 647 Mouse anti-Nestin (solid line histogram) or Alexa Fluor® 647 Mouse IgG1, k Isotype Control (dashed line histogram). The two peaks reveal differentiated neurons (low Nestin staining) and undifferentiated NSC (high Nestin staining). This antibody also works in BD Phosflow™ Perm Buffers II and III. Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
LEFT Image: Analysis of Alexa Fluor® 647 Mouse Anti-Nestin on human Neural Stem cells (NSCs). H9-derived NSCs were isolated by sorting from Embryoid bodies and were grown for 2 passages post sort, fixed (BD Cytofix™ Fixation Buffer, Cat. No. 554655) for 20 minutes at room temperature, permeabilized with BD Phosflow™ Perm Buffer I (Cat. No.557885), and then stained with either Alexa Fluor® 647 Mouse anti-Nestin (Cat. No. 560393; solid line histogram) or Alexa Fluor® 647 Mouse IgG1, κ Isotype Control (Cat. No. 557732; dashed line histogram). Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
RIGHT Image: Analysis of Alexa Fluor® 647 Mouse Anti-Nestin on human neurons. H9-derived NSC were differentiated into neurons for 12 days. The cells were fixed (BD Cytofix™ Fixation Buffer) for 20 minutes at room temperature, permeabilized with BD Phosflow™ Perm Buffer I, and then stained with either Alex Fluor® 647 Mouse anti-Nestin (solid line histogram) or Alexa Fluor® 647 Mouse IgG1, k Isotype Control (dashed line histogram). The two peaks reveal differentiated neurons (low Nestin staining) and undifferentiated NSC (high Nestin staining). This antibody also works in BD Phosflow™ Perm Buffers II and III. Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
Product Details
BD Pharmingen™
Rat (QC Testing), Human (Tested in Development)
Mouse IgG1, κ
Rat Nestin aa. 402-604 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645170
Aqueous buffered solution containing BSA, protein stabilizer, 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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
Product Notices
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- 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.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- 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
Perm Buffer III RUO
Size
125 ML
Cat No.
558050
Perm Buffer II RUO
Size
125 ML
Cat No.
558052
Fixation Buffer RUO
Size
100 ML
Cat No.
554655
Perm/Wash Buffer I RUO
Size
125 ML
Cat No.
557885
560393 Rev. 2
Antibody Details
25/NESTIN
The cytoskeleton consists primarily of core structural proteins that include microfilaments, microtubules, and intermediate filaments (IFs). IFs contain more than 50 distinct proteins that are organized into six different subtypes: Type I/II keratins expressed in epithelia, type III vimentin/desmin, type IV neurofilament proteins, type V nuclear lamins, and type VI nestin expressed primarily in embryonic cells. Nestin has a conserved core region (amino acids 7 to 314), which contains an α helical domain that is involved in coiled-coil assembly of IFs. The C-terminal region of nestin is similar to type IV IFs, since it contains highly charged amino acids, many glutamate residues, and an 11 amino acid repeat motif. Nestin is expressed in the cerebrum during embryonic development, in the cerebellum during early postnatal development, and in dermatomal cells and myoblasts during myogenesis. In vitro, nestin forms homodimers and homotetramers, but not IFs, and can co-assemble with type III vimentin and type IV internexin proteins. Thus, nestin is a core IF protein that is essential for proper cytoskeletal formation during neurogenesis and myogenesis.
560393 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 670-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
653 nm
669 nm
560393 Rev.2
Citations & References
View product citations for antibody "560393" on CiteAb
Development References (5)
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Jin K, Zhu Y, Sun Y, Mao XO, Xie L, Greenberg DA. Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc Natl Acad Sci U S A. 2002; 99(18):11946-11950. (Clone-specific: Immunofluorescence). View Reference
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Kachinsky AM, Dominov JA, Miller JB. Myogenesis and the intermediate filament protein, nestin. Dev Biol. 1994; 165(1):216-228. (Biology). View Reference
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Kernie SG, Erwin TM, Parada LF. Brain remodeling due to neuronal and astrocytic proliferation after controlled cortical injury in mice. J Neurosci Res. 2001; 66(3):317-326. (Clone-specific: Immunofluorescence). View Reference
-
Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell. 1990; 60(4):585-595. (Biology). View Reference
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Steinert PM, Chou YH, Prahlad V, et al. A high molecular weight intermediate filament-associated protein in BHK-21 cells is nestin, a type VI intermediate filament protein. Limited co-assembly in vitro to form heteropolymers with type III vimentin and type IV alpha-internexin. J Biol Chem. 1999; 274(14):9881-9890. (Biology). View Reference
560393 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.