Download A-10209A: Enumeration of Mitotic Cells with Dual

DRAFT: Rev. D
AN-10209A
APPLICATION INFORMATION
Flow Cytometry
ENUMERATION OF MITOTIC CELLS WITH DUAL
MEASUREMENTS OF HISTONE H3 PHOSPHORYLATION
AND DNA CONTENT
Yong Song, Mark Cheetham and Brendan S. Yee
Cellular Analysis Business Center, Beckman Coulter, Inc., Fullerton, CA 92835
Introduction
The cell cycle process can be described as a series
of distinct biochemical and morphological events
that occur in a reproducing cell population. The
distinct events within the cell cycle process can be
categorized into five main phases. Cells starting at
a resting state or G0, will proceed into the cell cycle
by entering Interphase (G1, S, G2) followed by the
M phase (mitosis and cytokinesis).1 As proliferating
cells transition from G1 to M, the DNA content will
double as the chromosomes are duplicated within
the cell.
DNA binding dyes such as 7-aminoactinomycin
D (7-AAD), Propidium Iodide or DAPI have been
commonly used in flow cytometry to measure the
change in DNA content of a cell population as it
transitions from G0, G1, S, G2 and M.
The use of a DNA binding dye alone can only
resolve the 5 Phases into 3 groups which are G0+G1,
S and G2+M. However if the % M phase is of
interest this technique is difficult to use, because
cells in the M phase and G2 have the same DNA
content.
In this application note, we describe an
enhanced method to differentiate cells that are
within the G2 and M phase in the cell cycle. This is
achieved by measuring the levels of histone H3
phosphorylation in each cell combined with the
DNA content utilizing a DNA binding dye, 7-AAD
(Beckman Coulter, Inc).
Histone H3 phosphorylation is highly correlated
with the G2 to M phase transition and chromosome
condensation. Histone H3 is phosphorylated at
Ser10 when cells enter the mitotic phase and
remains unphosphorylated in other phases of the
cell cycle.2
Anti-phospho-histone H3 (Ser 10) (Beckman
Coulter, Inc), is a rabbit polyclonal antibody
specific for histone H3 phoshorylated at serine 10.
This antibody is conjugated to Alexa Fluor® 488
which can be excited with a 488 nm laser. Its
peak emission is at 520 nm. Cell DNA content
measurement is carried out by 7-AAD staining
after cell permeabilization with methanol. 7-AAD
can also be excited with a 488 nm laser with an
emission peak from 650 to 675 nm.
Materials and methods:
• CC Standard L10 Polysterene Latex, (Beckman
Coulter PN 6602796)
• Anti-phospho-histone H3-Alexa Fluor 488,
(Beckman Coulter PN A24068)
• 7-aminoactinomycin D (7-AAD), (Beckman
Coulter PN IM3422)
Cell culture and treatment
Jurkat cells were cultured in RPMI-1640 supplement with 10% heat inactivated fetal bovine serum.
Cells at a concentration of 1 x 106 cells/mL were
treated with paclitaxel 2 µM) for 18-20 hours.
Double staining of phospho-histone H3
and DNA
Analysis Protocol Setup on the
Quanta SC software
The Electronic Volume (EV) is used to measure cell
size and can be calibrated using the CC Standard
L10 Polysterene Latex beads. Once the size scale
has been calibrated the entire cell population can
be brought on scale by adjusting the EV gain.
Debris can be gated out by adjusting the Lower
Level Discriminator (LLD).
If a larger amount of cell debris is generated
during cell treatment and sample preparation, data
collection can be triggered on FL3 (7-AAD). FL3
measurement is achieved by selecting linear display
(deselecting “Log” under “Parameter Info”) and
adjusting the FL3 PMT voltage to establish diploid
Go/G1 peak of samples at around channel 200. The
LLD is set to a level where the noise signal is not
visible or just slightly visible on the graph.
For Alexa Fluor 488 (FL1) measurement, select
the “Log” display under “Parameter Info” and
adjust the FL1 PMT voltage to locate the FL1
population of control or treated cell completely on
scale. The FL1 negative cells should be on the edge
of the first log decade population and, this can be
confirmed with the isotypic control.
The phospho-histone H3 positive events can
be enumerated by creating a polygon or ellipse
region on the FL1 and FL3 dual parameter graph
(Figure 1 and 2).
Follow the product insert of Anti- phospho-histone
H3-Alexa Fluor 488 for cell fixation, permeabilization and staining. Alternatively, the following
staining protocol can be used.
1. Fixation and permeabilization
• Accurately determine cell count with the Cell
Lab Quanta™ SC, Z2™ COULTER
COUNTER® or Vi-CELL® Series Cell
Viability Analyzer
• Aliquot 1 x 106 cells into a 1.5 or 2 mL assay
tube
• Centrifuge at 300 g for 5 min at 4°C and
aspirate supernatant
• Wash cells with 1 mL of PBS
• Fix and permeabilize cells by adding 1 mL
ice-cold 100% methanol slowly to the cells
while gently vortexing.
• Incubate 10 min on ice
• Spin at 300 g for 10 min at room temperature
and aspirate supernatant
2. Staining
• Wash cells once with 1 mL of PBS + 0.5%
BSA
• Remove supernatant and resuspend cells with
100 µL of PBS + 0.5% BSA
• Add 10 µL of phospho-histone H3-Alexa
Fluor 488 or the isotypic control
• Mix gently and incubate 60 min at 20-25°C
in dark
• Wash cells twice with 1.5 mL of PBS + 0.5%
BSA
• Resuspend cells with 100 µL of PBS + 0.5%
BSA
• Add 20 µL of 7-AAD
• Mix gently and incubate 5-10 min at 20-25oC
in dark
• Add 400 µL of PBS + 0.5% BSA
• Analyze on the Quanta SC
Filter configuration
Use the standard laser 488nm filter configuration
for the Cell Lab Quanta.
• 550 DLP
• z488 RDC
• 525/40 BP (FL1)
• 600 DLP
• 670LP (FL3)
2
Figure 1: Untreated control Jurkat cells stained with Anti-phosphohistone H3 – Alexa Fluor 488 (pHis) and 7-AAD. Phospho-histon
H3 positive cells are circled in blue.
Figure 2: Paclitaxel (2 µM) treated Jurkat cells stained with Antiphospho-histone H3-Alexa Fluor 488 and 7-AAD. As can be seen
by comparing Figures 1 and 2, the paclitaxel treated sample had a
larger proportion of cells in M phase (27 % in the treated and 2%
in the untreated), indicating a cell cycle arrest has occurred
3
Bibliography
1. Enten J, Monson M. DNA cell cycle analysis
with 4’,6-diamidino-2-phenylindole (DAPI) or
propidium iodide (PI) nuclear stains. Cell Lab
Technical Application Information Bulletins 2004;
http://www.beckmancoulter.com/literature/Biores
earch/A-2012A.pdf
2. Hendzel MJ, Wei Y, Mancini, MA, Ranalli T,
Brinkley BR, Bazett-Jones DP, and Allis CD.
Mitosis-specific phosphorylation of histone H3
initiates in centromeric heterochromatin and
spreads in an ordered fashion coincident with
mitotic chromosome condensation. Chromosoma
1997; 106:348-360
Alexa Fluor® is a registered trademark of Invitrogen Corporation
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© 2006 Beckman Coulter, Inc.
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