Flow cytometric analysis of H2AX (pS139) expression in human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells were either not treated (dashed line histogram) or cultured with 50 μM Etoposide (Calbiochem, Cat. No. 341205) for 2 hrs at 37°C (solid line histogram). The cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No.558050). After washing with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), the cells were stained with PerCP-Cy™5.5 Mouse Anti-H2AX (pS139) antibody (Cat. No. 564718). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Flow cytometric analysis of H2AX (pS139) expression in human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells were either not treated (dashed line histogram) or cultured with 50 μM Etoposide (Calbiochem, Cat. No. 341205) for 2 hrs at 37°C (solid line histogram). The cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No.558050). After washing with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), the cells were stained with PerCP-Cy™5.5 Mouse Anti-H2AX (pS139) antibody (Cat. No. 564718). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Product Details
BD Pharmingen™
H2A.X; H2A/X; H2AFX; HIST5-2AX; gamma-H2AX; γ-H2AX; H2AX (pS140)
Human (QC Testing), Mouse (Tested in Development)
Mouse BALB/c IgG1, κ
Phosphorylated Human H2AX Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2738912
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 PerCP-Cy5.5 under optimum conditions, and unconjugated antibody and free PerCP-Cy5.5 were removed. Storage of PerCP-Cy5.5 conjugates in unoptimized diluent is not recommended and may result in loss of signal intensity.
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).
- 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.
- Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
- PerCP-Cy5.5–labelled antibodies can be used with FITC- and R-PE–labelled reagents in single-laser flow cytometers with no significant spectral overlap of PerCP-Cy5.5, FITC, and R-PE fluorescence.
- PerCP-Cy5.5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using dual-laser cytometers, which may directly excite both PerCP and Cy5.5™. We recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Cy is a trademark of GE Healthcare.
- 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
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm Buffer III RUO
Size
125 mL
Cat No.
558050
564718 Rev. 2
Antibody Details
N1-431
Histones are highly basic proteins that complex with DNA to form chromatin. The H2AX histone (~15 kDa calculated molecular weight) is a member of the H2A histone family whose members are components of nucleosomal histone octamers. Double-stranded breaks in DNA caused by replication errors, apoptosis, or other physiological processes (including, immunoglobulin and TCR gene recombinations) and DNA damage caused by ionizing radiation, UV light, or cytotoxic agents lead to phosphorylation of H2AX on serine 139. H2AX (pS139) is also referred to as H2AX (pS140) when the N-terminal methionine that is normally excised during posttranslational processing is included in amino acid sequence numbering. Kinases such as ataxia telangiectasia mutated (ATM) or ATM-Rad3-related (ATR) phosphorylate H2AX to induce its function. Phosphorylated H2AX (also termed, gamma-H2AX) functions to recruit and localize DNA repair proteins or cell cycle checkpoint factors to the DNA-damaged sites. In this way, phosphorylated H2AX promotes DNA repair and maintains genomic stability and thus helps prevent oncogenic transformations.
564718 Rev. 2
Format Details
PerCP-Cy5.5
PerCP-Cy5.5 dye is part of the BD blue family of dyes. This tandem fluorochrome is comprised of a fluorescent protein complex (PerCP) with an excitation maximum (Ex Max) of 482 nm and an acceptor dye with an emission maximum (Em Max) at 676 nm. PerCP-Cy5 is designed to be excited by the blue laser (488-nm) and detected using an optical filter centered near 680 nm (e.g., a 695/40 nm bandpass filter). The donor dye can be partially excited by the Violet (405-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
PerCP-Cy5.5
Blue 488 nm
482 nm
676 nm
564718 Rev.2
Citations & References
Development References (7)
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Austin WR, Armijo AL, Campbell DO, et al.. Nucleoside salvage pathway kinases regulate hematopoiesis by linking nucleotide metabolism with replication stress. J Exp Med. 2012; 209(12):2215-2228. (Clone-specific: Flow cytometry). View Reference
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Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem. 2001; 276(45):42462-42467. (Biology). View Reference
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Fernandez-Capetillo O, Lee A, Nussenzweig M, Nussenzweig A. H2AX: the histone guardian of the genome. DNA Repair (Amst). 2004; 3(8-9):959-967. (Biology). View Reference
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Kuo LJ, Yang LX. Gamma-H2AX - A novel biomarker for DNA double-strand breaks. In Vivo. 2008; 22(3):305-309. (Biology). View Reference
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Lavelle D, Vaitkus K, Ling Y, et al. Effects of tetrahydrouridine on pharmacokinetics and pharmacodynamics of oral decitabine. Blood. 2012; 119(5):1240-1247. (Clone-specific: Flow cytometry). View Reference
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Rogakou EP, Nieves-Neira W, Boon C, Pommier Y, Bonner WM. Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. J Biol Chem. 2000; 275(13):9390-9395. (Biology). View Reference
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Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998; 273(10):5858-5868. (Biology). View Reference
564718 Rev. 2
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
