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Alexa Fluor® 488 Mouse anti-H2AX (pS139)
Alexa Fluor® 488 Mouse anti-H2AX (pS139)

Immunofluorescent staining of human cell line. HeLa cells (ATCC CCL-2) were seeded in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) at ~10,000 cells per well. After overnight culture, the cells were exposed to 2400 Joules UV irradiation (right image) or untreated (left image) and then allowed to recover for 30-60 minutes at 37°C. The cells were fixed, permeabilized with cold methanol, and stained with Alexa Fluor® 488 Mouse anti-H2AX (pS139) (pseudo colored green) according to the Recommended Assay Procedure. Cell nuclei were counterstained with Hoechst 33342 (pseudo colored blue). The images were captured on a BD Pathway™ 435 high-content Bioimager system using a 20X objective and merged using BD AttoVision™ software. This antibody also worked with the Saponin and the Triton™ X-100 Perm/Wash protocols (see Recommended Assay Procedure; Bioimaging protocol link).

Alexa Fluor® 488 Mouse anti-H2AX (pS139)

Analysis of H2AX (pS139) in human peripheral blood mononuclear cells (PBMC). PBMC were either stimulated with 50 μM Etoposide (Calbiochem, Cat. No. 341205 ) for 4hrs at 37°C (shaded histogram) or untreated (open histogram). The cells were fixed and permeabilized using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (Cat. No. 554714) and then stained with Alexa Fluor® 488 Mouse anti-H2AX (pS139). Flow cytometry was performed on a BD™ LSR II system.

Immunofluorescent staining of human cell line. HeLa cells (ATCC CCL-2) were seeded in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) at ~10,000 cells per well. After overnight culture, the cells were exposed to 2400 Joules UV irradiation (right image) or untreated (left image) and then allowed to recover for 30-60 minutes at 37°C. The cells were fixed, permeabilized with cold methanol, and stained with Alexa Fluor® 488 Mouse anti-H2AX (pS139) (pseudo colored green) according to the Recommended Assay Procedure. Cell nuclei were counterstained with Hoechst 33342 (pseudo colored blue). The images were captured on a BD Pathway™ 435 high-content Bioimager system using a 20X objective and merged using BD AttoVision™ software. This antibody also worked with the Saponin and the Triton™ X-100 Perm/Wash protocols (see Recommended Assay Procedure; Bioimaging protocol link).

Analysis of H2AX (pS139) in human peripheral blood mononuclear cells (PBMC). PBMC were either stimulated with 50 μM Etoposide (Calbiochem, Cat. No. 341205 ) for 4hrs at 37°C (shaded histogram) or untreated (open histogram). The cells were fixed and permeabilized using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (Cat. No. 554714) and then stained with Alexa Fluor® 488 Mouse anti-H2AX (pS139). Flow cytometry was performed on a BD™ LSR II system.

Product Details
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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
Bioimaging (Routinely Tested), Intracellular staining (flow cytometry) (Tested During Development)
5 µl
AB_1645352
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® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was removed.

Recommended Assay Procedures

Recommended Assay Procedure for Bioimaging:

http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp or  http://www.bdbiosciences.com/bioimaging/reagents

1.        Seed the cells in appropriate culture medium at an appropriate cell density in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219), and

        culture overnight to 48 hours.

2.        Remove the culture medium from the wells, and wash (one to two times) with 100 μl of 1× PBS.

3.        Fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or BD Cytofix™ fixation buffer (Cat. No. 554655) to each well and incubating for 10 minutes at room temperature (RT).

4.        Remove the fixative from the wells, and wash the wells (one to two times) with 100 μl of 1× PBS.

5.        Permeabilize the cells using either cold methanol (a), Triton™ X-100 (b), or Saponin (c):

a.        Add 100 µl of -20°C 90% methanol or -20°C BD™ Phosflow Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.

b.        Add 100 µl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.

c.        Add 100 µl of 1× Perm/Wash buffer (Cat. No. 554723) to each well and incubate for 15 to 30 minutes at RT.  Continue to use 1× Perm/Wash buffer for all subsequent wash and dilutions steps.

6.        Remove the permeabilization buffer from the wells, and wash one to two times with 100 μl of appropriate buffer (either 1× PBS or 1× Perm/Wash buffer, see step 5.c.).

7.        Optional blocking step:  Remove the wash buffers, and block the cells by adding 100 µl of blocking buffer BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) or 3% FBS in appropriate dilution buffer to each well and incubating for 15 to 30 minutes at RT.

8.        Dilute the antibody to its optimal working concentration in appropriate dilution buffer. Titrate purified (unconjugated) antibodies and second-step reagents to determine the optimal concentration.  If using a Bioimaging Certified antibody conjugate, dilute it 1:10.

9.        Add 50 µl of diluted antibody per well and incubate for 60 minutes at RT.  Incubate in the dark if using fluorescently labeled antibodies.

10.        Remove the antibody, and wash the wells three times with 100 μl of wash buffer.  An optional detergent wash (100 μl of 0.05% Tween in 1× PBS) can be included prior to the regular wash steps.

11.   If the antibody being used is fluorescently labeled, then move to step 12.  Otherwise, if using a purified unlabeled antibody, repeat steps 8 to 10 with a fluorescently labeled second-step reagent to detect the purified antibody.

12.        After the final wash, counter-stain the nuclei by adding 100 μl of a 2 μg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.

13.        View and analyze the cells on an appropriate imaging instrument.  Recommended filters for the BD Pathway™ instruments are:

Instrument                        Excitation        Emission        Dichroic

BD Pathway 855                488/10                515 LP                Fura/FITC

BD Pathway 435                482/35                536/40                FF506

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test when following the Recommended Assay Procedure. A Test is typically ~10,000 cells cultured in a well of a 96-well imaging plate.
  2. 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.
  3. Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. 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.
  6. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  7. Triton is a trademark of the Dow Chemical Company.
  8. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  9. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560445 Rev. 2
Antibody Details
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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. Immunofluorescent staining and bioimaging analysis of cultured cells can be used to readily identify H2AX (pS139)-containing foci. As such, H2AX (pS139) immunofluorescence localization serves as a biomarker for nuclear sites of DNA damage (e.g., double-stranded DNA breaks) in affected cells.

560445 Rev. 2
Format Details
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Alexa Fluor™ 488
Alexa Fluor™ 488 Dye is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 517-nm. Alexa Fluor™ 488 is designed to be excited by the Blue laser (488 nm) and detected using an optical filter centered near 520-nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 488
Blue 488 nm
494 nm
517 nm
560445 Rev.2
Citations & References
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Development References (5)

  1. 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
  2. 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
  3. Kuo LJ, Yang LX. Gamma-H2AX - A novel biomarker for DNA double-strand breaks. In Vivo. 2008; 22(3):305-309. (Biology). View Reference
  4. 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
  5. 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
View All (5) View Less
560445 Rev. 2

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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.