Product Datasheet
Mitotic Index Flow Cytometry Assay: Histone H3
pSer10 + Propidium Iodide ab151282
4 Images
Overview
Product name
Mitotic Index Flow Cytometry Assay: Histone H3 pSer10 + Propidium Iodide
Detection method
Fluorescent
Sample type
Adherent cells, Suspension cells
Assay type
Quantitative
Assay time
2h 00m
Species reactivity
Reacts with: Mouse, Rat, Human
Product overview
ab151282 is a flow cytometry assay designed for quantitative mitotic index and DNA content analysis using an
anti-HistoneH3 pSer10 antibody and the nucleic acid stain Propidium iodide.
The anti-HistoneH3 pSer10 antibody is a rabbit monoclonal directly conjugated to the fluorescent dye Alexa
Fluor® 488 that specifically labels mitotic cells. Propidium iodide staining of DNA is the classic means of cell
cycle analysis by DNA content. The staining procedure takes less than 2 hours and the contents of this kit are
sufficient for 100 assays.
Notes
The DNA of mammalian, yeast, plant or bacterial cells can be stained by a variety of DNA binding dyes. The
premise with these dyes is that they are stoichiometric i.e. they bind in proportion to the amount of DNA present
in the cell. In this way cells that are in S phase will have more DNA than cells in G1. They will take up
proportionally more dye and will fluoresce more brightly until they have doubled their DNA content. The cells in G2
will be approximately twice as bright as the cells in G1. Propidium iodide is a fluorescent molecule that binds
nucleic acid with little or no sequence preference. Because Propidium iodide binds RNA as well as DNA, RNaseA
(ribonuclease A) is included in this kit to digest cellular RNA and thus decrease background RNA staining from
the experiment.While Propidium iodide will reveal the DNA content of a cell, it does not provide molecular details
underlying the DNA content, such as what percentage are actually mitotic.
Histone H3 is one of the four core histone proteins (H2A, H2B, H3 and H4) that pack DNA in nucleosomes.
Phosphorylation of Histone H3 at Ser10 is tightly correlated with chromosome condensation during mitosis.
Hence, Histone H3 pSer10 signal indicates a mitotic cell with condensed DNA. The anti-Histone H3 pSer10
antibody in this kit is a rabbit monoclonal antibody directly labeled with the fluorescent dye Alexa Fluor® 488.
Tested applications
Flow Cyt
Platform
Reagents
Properties
Storage instructions
Store at +4°C. Please refer to protocols.
Components
100 tests
100X Triton X-100
1 x 1.25ml
10X Blocking Buffer
1 x 20ml
10X Phosphate Buffered Saline
1 x 100ml
200X RNaseA
1 x 100µl
20X Propidium Iodide
1 x 2ml
500X anti-HistoneH3 pSer10 Alexa Fluor ® 488 conjugate
1 x 20µl
Relevance
Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility
to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription
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Product Datasheet
regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex
set of post-translational modifications of histones, also called histone code, and nucleosome remodeling.
Cellular localization
Nucleus. Chromosome.
Applications
Our Abpromise guarantee covers the use of ab151282 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application
Abreviews
Flow Cyt
Notes
Use at an assay dependent concentration.
Mitotic Index Flow Cytometry Assay: Histone H3 pSer10 + Propidium Iodide images
Asynchronous and treated Hela cells were analyzed for
mitotic cells. Thymidine treatment (2 mM, 24h) enriches
for G1/S arrested cells and nocodazole treatment (100
ng/mL, 24h) enriches for mitotic cells. The R1 box
indicates cells that stain positively for HistoneH3 pSer10.
Relative to asynchronous cells (3.6%), thymidine
Sample analysis of an experiment using ab151282
treatment reduces the number of HistoneH3 pSer10
on HeLa cells.
positive cells (0.8%) whereas nocodazole greatly
increases the number of positive cells (76.7%).
Asynchronous Jurkat cells were harvested and the antiHistoneH3 pSer10 Alexa Fluor® 488 antibody was either
omitted (left) or included at 1X (right). Both samples were
stained with Propidium iodide. Propidium iodide is plotted
on the X-axis (linear scale) and HistoneH3 pSer10 Alexa
Fluor® 488 is on the Y axis (log scale). The R1 box
indicates cells that stain positively for HistoneH3 pSer10.
In the right panel, note that only cells with 4N DNA content
Sample flow cytometry data using ab151282 on
untreated Jurkat cells.
have Histone H3 pSer10 staining. This result indicates
that 2.9% of the asychronous cell population is
undergoing mitosis. There is no staining when the
antibody is omitted (left).
HeLa cells were stained with anti-HistoneH3 pSer10
Alexa Fluor® 488 antibody (green) and DAPI (nuclei, blue).
The anti-histoneH3 pSer10 antibody clearly labels
condensed DNA chromosomes.
Immunocytochemistry validation of the HistoneH3
pSer10 Alexa Fluor® 488 primary antibody used in
ab151282.
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Product Datasheet
Asynchronous HeLa cells were stained with antiHistoneH3 pSer10 Alexa Fluor® 488 antibody (green) and
DAPI (nuclei, blue). Note that only mitotic and pre-mitotic
cells with condensed nuclei (based on DAPI stain) have
HistoneH3 pSer10 staining.
Immunocytochemistry validation of the HistoneH3
pSer10 Alexa Fluor® 488 primary antibody used in
ab151282.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
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