ab151282 –Mitotic Index Flow Cytometry Assay: Histone H3 pSer10 + Propidium Iodide

Instructions for Use

To simultaneously determine HistoneH3 pSer10 and DNA content using a flow cytometer. Human, mouse and rat reactive.

This product is for research use only and is not intended for diagnostic use.

Table of Contents

1. Introduction 2

2. Assay Summary 4

3. Kit Contents 5

4. Storage and Handling 5

5. Additional Materials Required 5

6. Preparation of Reagents 6

7. Sample Preparation 7

8. Assay Procedure 9

9. Data Analysis 11

10. Assay Performance 13

11. Frequently Asked Questions 16

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1. Introduction

Principle: 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.

Background: Cell cycle analysis by quantitation of DNA content

was one of the earliest applications of flow cytometry. 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

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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. For

example, Propidium iodine staining can determine the portion of cells

that have 4N DNA content, but does not reveal what percentage of

the those 4N cells have just completed DNA synthesis and 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.

This kit is compatible with human, mouse and rat cells that can be

prepared as a single cell suspension. A flow cytometer is required

for quantitative analysis.

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2. Assay Summary

Culture cell line(s) of interest. Perform treatment(s) of interest.

Harvest cells in single cell suspension;

fix with 4% Paraformaldehyde

Block and permeabilize cells.

Stain with anti-HistoneH3 pSer10 Alexa Fluor® 488 antibody:

45min.

Stain cells with Propidium Iodide + RNase: 30min

Analyze on flow cytometer:

HistoneH3pSer10-Alexa Fluor ® 488: collect on

FL1[Ex/Em=495/519nm]

Propidium Iodide: collect on FL2 [Ex/Em=493/636nm]

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3. Kit Contents

Item Quantity

10X Phosphate Buffered Saline (PBS) 100 mL

10X Blocking Buffer 20 mL

100X Triton X-100 1.25 mL

500X anti-HistoneH3 pSer10 Alexa Fluor ® 488 conjugate

200 µL

20X Propidium iodide (1 mg/mL) 2 mL

200X RNaseA (110,000 U/mL) 100 µL

4. Storage and Handling

This kit is shipped at +4°C. Upon receipt, store the kit at +4°C.

5. Additional Materials Required

Nanopure water or equivalent.

Paraformaldehyde

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6. Preparation of Reagents

6.1 Prepare an appropriate amount of 1X PBS in nanopure

water or equivalent. For example, dilute 5 mL of 10X PBS

in 45 mL water. Mix well and store at room temperature.

6.2 Immediately prior to use prepare the 1X Blocking Solution

by diluting 10X Blocking Solution in 1X PBS. For example,

dilute 2 mL 10X Block Solution in 18mL 1X PBS.

6.3 Immediately prior to use prepare 1X Permeabilization

Solution by diluting 100X Triton X-100 to 1X in 1X Blocking

Solution. For example: 20 µL 100X Triton X-100 to 1.98mL

1X Blocking Solution.

6.4 Immediately prior to use prepare 1X anti-HistoneH3 pSer10

Alexa Fluor® 488 antibody staining solution in 1X Blocking

Solution. For example, add 2 µL 500X anti-HistoneH3

pSer10-Alexa Fluor® 488 to 1 mL 1X Blocking Solution.

6.5 Immediately prior to use prepare the Propidium Iodide +

RNase staining solution in PBS. For example, 9.45 mL

PBS + 500 µL 20X Propidium Iodide + 50 µL 200X RNase.

Note – Propidium iodide is toxic! Handle with care and

dispose of according to local regulations.

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7. Sample Preparation

A critical component of any flow cytometry experiment is having a single-cell suspension of the cells being analyzed. Means of achieving single-cell suspensions from adherent cells and suspension cells are detailed below.

The anti-histoneH3 pSer10 antibody in this assay is reactive with human, mouse and rat samples. Propidium iodide will stain the DNA of all species.

The volumes given below assume a 6-well plate equivalent of cells (~100,000 – 600,000 cells). Volumes can be scaled-up proportionately when processing more cells.

At no point should the cells or cell pellets be allowed to dry.

Two methods exist for collecting adherent and suspension cell.

Adherent Cells

7.1 Culture cells normally.

7.2 Generate a single cell suspension in a manner similar to

routine passaging of the cells: Remove the culture media,

rinse the cells with PBS and add trypsin to dissociate cells.

Importantly, do not discard the culture media or PBS rinse

as they likely contain loosely adherent cells (mitotic or

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apoptotic) that should be included in analysis. Instead,

pool and save the culture media and PBS rinse.

7.3 Once cells are fully trypsinized (detached from plate and

from each other), quench the media with the saved media

+ PBS wash from the previous step. Gently pipette up and

down if necessary to generate a single cell suspension.

Suspension Cells7.1 Culture cells normally.

7.2 If necessary, gently pipette cells up and down so disperse

any cell aggregates.

7.3 Proceed to step 7.4 below.

7.4 Pellet the cells at 500 x g for 5 minutes and aspirate and

discard the supernatant.

7.5 Optional: Wash cells with 1X PBS: Add 1 mL 1X PBS to the

cell pellet, gently resuspend the cell pellet, centrifuge again

at 500 x g for 5 minutes.

7.6 Fix the cells in 4% paraformaldehyde: gently resuspend the

cells in 400 µL PBS. Add 100 µL 20% paraformaldehyde

and mix gently. Incubate for 10 minutes.

7.7 Pellet the cells at 500 x g for 5 minutes and remove the 4%

paraformaldehyde supernatant.

Note – Paraformaldehyde is toxic. Handle with care and dispose of according to local regulations.

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7.8 Wash cells with 1X PBS: Add 1 mL 1X PBS to the cell

pellet, gently resuspend the cell pellet, centrifuge again at

500 x g for 5 minutes.

7.9 This is a potential stopping point in the assay: one can

continue to the staining portion of the assay or store the

fixed cells at 4ºC. If storing cells, resuspend in 500 µL PBS

with 0.02% sodium azide and store in the refrigerator. If

proceeding, move directly to step 8.4 below.

8. Assay Procedure

The following antibody and Propidium iodide staining procedure should be performed immediately before analysis on the flow cytometer. Cells are first stained with antibody followed by Propidium iodide staining. Note that Propidium iodide stains DNA in equilibrium with the buffer: cells are analyzed in the Propidium Iodide staining mixture (the dye is not washed away from the cells).

The volumes given below assume ~100,000 – 500,000 cells per tube. Volumes can be adjusted up or down proportionately with cell number.

The entire procedure should be completed at room temperature.

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8.1 Transfer the previously prepared cells from 4ºC to the

benchtop and equilibrate to room temperature.

8.2 Gently resuspend cells by inverting the tube or by gentle

up and down pipetting.

8.3 Pellet the cells at 500 x g for 5 minutes. Carefully aspirate

the supernatant without disrupting the pellet. It is better to

incompletely remove the supernatant than to accidently

aspirate part of the pellet and lose cells.

8.4 Block and permeabilize the cells by resuspending in 200

µL 1X Permeabilization solution. Incubate 5 minutes at

room temperature.

8.5 Pellet the cells at 500 x g for 5 minutes. Carefully aspirate

the supernatant without disrupting the pellet.

8.6 Add 100 µL 1X anti-HistoneH3 pSer10 Alexa Fluor® 488.

Make sure that the cells are resuspended in the antibody

solution and incubate in the dark for 45 minutes.

For the rest of the protocol samples should be kept in the

dark.

8.7 Wash away unbound antibody: gently add 1 mL 1X

blocking solution to the sample and pellet at 500 x g for 5

minutes. Aspirate and discard the supernatant.

8.8 Gently resuspend the cell pellet in 200 µL 1X Propidium

iodide + RNase staining solution (see 6.5). Make certain

that the cells are fully resuspended.

Note – Propidium Iodide is toxic. Handle with care and dispose of according to local regulations.

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8.9 Incubate in the dark for 30 minutes.

8.10 During the incubation prepare equipment as necessary.

8.11 Gently resuspend cells that settled during the incubation

by pipetting up and down. If desired, pass cells through

an appropriate filter (not provided) to remove cell

aggregates.

8.12 Run samples on flow cytometer: Set appropriate FSC vs.

SSC gates to exclude debris and cell aggregates. Collect

HistoneH3 pSer10 fluorescence in FL1. Collect Propidium

iodide fluorescence in FL2.

HistoneH3pSer10 Alexa Fluor® 488: collect on FL1[Ex/Em=495/519nm]

Propidium Iodide: collect on FL2 [Ex/Em=493/636nm]

9. Data Collection and Analysis

The following are general guidelines. Specific methods of analysis

will vary with different flow cytometer analysis programs.

9.1 Establish appropriate FSC vs. SSC gates to exclude

debris and cell aggregates.

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9.2 Collect the HistoneH3 pSer10 Alexa Fluor® 488

fluorescence in the appropriate channel (typically FL1)

using 488nm laser illumination.

9.3 Collect Propidium iodide fluorescence in the appropriate

channel (typically FL2) using 488nm laser illumination.

9.4 Expect to see a 2-fold intensity difference between the 2N

and 4N Propidium iodide peaks.

9.5 Expect to see the HistoneH3 pSer10 Alexa Fluor® 488

positive cells to be the 4N DNA content cells, as in Figures

1 and 2.

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10. Assay Performance

Figures 1 and 2 below show typical results using ab151282 on HeLa

and Jurkat cell lines. A useful way to analyze the data is to plot

Propidium iodide intensity on the x-axis and HistoneH3 pSer10

intensity on the y-axis.

Figures 3 and 4 are immunocytochemistry data using the HistoneH3

pSer10 Alexa Fluor® 488 antibody in ab151282. This data

demonstrates the specificity of this antibody for labeling pre-mitotic

and mitotic cells with condensed DNA.

Figure 1. Sample flow cytometry data using ab151282 on untreated Jurkat cells. Asynchronous Jurkat cells were harvested

and the anti-HistoneH3 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

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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 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).

Figure 2. Sample analysis of an experiment using ab151282 on HeLa cells. 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 treatment reduces the number of HistoneH3 pSer10

positive cells (0.8%) whereas nocodazole greatly increases the

number of positive cells (76.7%).

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Figure 3. Immunocytochemistry validation of the HistoneH3 pSer10 Alexa Fluor® 488 primary antibody used in ab151282. Asynchronous HeLa cells were stained with anti-HistoneH3 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.

Figure 4. Immunocytochemistry validation of the HistoneH3 pSer10 Alexa Fluor® 488 primary antibody used in ab151282. 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.

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11. Frequently Asked Questions

11.1 How many cells do I need to analyze?

A general guideline is to analyze 10,000 cells per sample.

Generally there is some cell loss during the fixation and

staining procedure, so it is recommended to harvest at least

10 times that many cells. The assay will perform well in the

range of 500,000 – 2,500,000 cells/mL in the Propidium

iodide + RNase staining solution. Adjust the volume of the

Propidium Iodide + RNase staining solution appropriately to

achieve this concentration range

11.2 What is the relationship between DNA content and cell

cycle?

Cells with two copies of each chromosome (2N) are not

actively in the process of cell replication and are in one of

two states: G1 (not currently replicating, but retain the ability

to replicate) or G0 (senescent). Cells that are in the process

of replicating and are actively copying their DNA have an

amount of DNA that is intermediate between 2N and 4N (2N

– 4N). After DNA synthesis is completed the DNA content is

doubled (4N) and cells are either in a resting state (G2) or in

mitosis (M). For more information see product ab139418

and find the cell cycle resource at www.abcam.com.

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11.3 Is there an alternative to paraformaldehyde fixation?

It is possible to fix cells with ethanol instead of

paraformaldehyde. Often there are tradeoffs between

antibody staining and propidium iodide staining when

selecting a fixation method. For this kit, we recommend

paraformaldehyde fixation because the antibody staining is

superior compared to ethanol fixed cells. However, the

propidium iodide staining is superior in ethanol fixed cells –

2N and 4N DNA content peaks are sharper and better

resolved. It is up to the user to evaluate if maximum

resolution is required for the antibody or propidium iodide

data.

For details on fixing cells in 66% ethanol, see the detailed

protocol in ab139418.

It is also possible to use the contents of this kit to stain

samples in parallel – one sample using paraformaldehyde

fixed cells for the HistoneH3 pSer10 staining and a parallel

sample fixed in ethanol for the propidium iodide stain.

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The Alexa Fluor® dye included in this product is provided under an intellectual property license from Life Technologies Corporation. As this product contains the Alexa Fluor® dye, the purchase of this product conveys to the buyer the non-transferable right to use the purchased product and components of the product only in research conducted by the buyer (whether the buyer is an academic or for-profit entity). As this product contains the Alexa Fluor® dye the sale of this product is expressly conditioned on the buyer not using the product or its components, or any materials made using the product or its components, in any activity to generate revenue, which may include, but is not limited to use of the product or its components: (i) in manufacturing; (ii) to provide a service, information, or data in return for payment (iii) for therapeutic, diagnostic or prophylactic purposes; or (iv) for resale, regardless of whether they are sold for use in research. For information on purchasing a license to use products containing Alexa Fluor® dyes for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.

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