A-2012A
AAPPLICATION
PPLICATION IINFORMATION
NFORMATION
Flow Cytometry
DNA CELL CYCLE ANALYSIS WITH 4′, 6-DIAMIDINO-2PHENYLINDOLE (DAPI) OR PROPIDIUM IODIDE (PI)
NUCLEAR STAINS
Jay Enten, MaryLyn Monson
Cellular Analysis Business Center, Beckman Coulter, Inc., Miami, FL 33196
Introduction
Growth and proliferation in eukaryotic cells is characterized with distinct phases of development known
as the cell cycle.
The sequence of cell cycle events can be identified as follows: Initiating from a quiescent or resting
state (Phase G0), cell growth and preparation of
chromosomes for replication takes place (Phase G1).
The cycle continues with synthesis of DNA (S Phase)
and is followed by preparations for cell division
(Phase G2). The cycle completes with mitosis (M
Phase) and is perpetuated with the newly divided
cells.
Flow cytometry offers a rapid method for measuring the DNA content of cells and provides a convenient research tool to monitor cell cycle status and
regulation. An exponentially growing population of
cells will have a DNA content distribution containing
an initial peak of G0/G1 cells, a valley of S Phase
cells, and a second peak containing G2/M cells. Cells
in the G2/M Phase have twice the DNA content as
cells in the G0/G1 Phase (see Figure 4).
† Contains LPR (Lysing and Permeabilizing reagent) and
Propidium Iodide stain.
DNA content measurements can be performed
with the Cell Lab Quanta,™ and are based on the
ability of nuclear dyes, such as DAPI and Propidium
Iodide, to bind selectively to DNA under appropriate
staining conditions. Cells stained with such dyes
emit fluorescence in direct proportion to their DNA
content.
The Cell Lab Quanta is a flow cytometry system
designed to simultaneously measure Electronic Cell
Volume (EV) and fluorescence. In this application
note, methods for cell cycle analysis are described
which can measure fluorescence from stained cells
using either a Mercury Arc Lamp and DAPI, or a
488 nm Laser and Propidium Iodide.
Materials
• Flow-Check™ Fluorospheres PN 6605359
• DNA Prep Reagent Kit† PN 6607055
• NIM-DAPI 10 µg/mL PN 731085
• Filter Tips PN 731087
• Single cell suspensions or Fresh/Frozen Tissue
• Phosphate Buffered Saline (PBS)
• Trout Red Blood Cells (TRBC) PN 629972
(DNA Reference Calibrator)
Procedure
Instrument Configuration
NIM-DAPI Methodology
DAPI (see Figure 1.)
Cells in Suspension
• Mercury Arc Lamp - 365 nm
• UG1 Exciter
• 560 Short Pass Dichroic
• 450/55 Band Pass (FL1)
• 570 Long Pass (FL2)
1. Add 1 drop of TRBC directly to 1 mL of
NIM-DAPI.
2. Add single cell suspensions directly to 1 mL of
NIM-DAPI to obtain a final cell concentration
of 1 - 2 x 106 cells/mL.
3. Filter all samples with a ~25 µm filter tip into a
sample cup and analyze.
Propidium Iodide (see Figure 2.)
• 488 nm Laser - 20 mW
• 560 Short Pass Dichroic
• 525/40 Band Pass (FL1)
• 570 Long Pass (FL2)
Solid Tissue Specimens
1. Place fresh or frozen tissue in a mincing dish
containing 2 mL of NIM-DAPI.
2. Shred the tissue until it is completely mixed in
the solution. Obtain a final cell concentration of
1 - 2 x 106 cells/mL.
3. Filter with a ~25 µm filter tip into a sample cup
and analyze.
Selected References
1. Raber M.N. and Barlogie R.: 1990. DNA Flow
Cytometry of Human Solid Tumors. In: Flow
Cytometry and Sorting. 2nd ed., New York, NY:
Wiley-Liss, Inc, p. 745-754.
2. Krishan A., Ganapathi R.N. and Israel M.:
1978. Effect of adriamycin and analogs on the
nuclear fluorescence of Propidium iodidestained cells. Cancer Res. 38: 3656-3662.
3. Shapiro H.M.: 1995. Practical Flow Cytometry,
3rd ed. New York, NY. Wiley-Liss, p. 198-199,
266-267, 372-373.
4. Vindelov L.L., Christensen I.J. and Nissen N.I.:
1983. Standardization of high-resolution flow
Cytometric DNA analysis by the simultaneous
use of chicken and trout red blood cells as internal reference standards. Cytometry 3: 328-331.
5. Ormerod M.G.: 1994. Analysis of DNA general
methods in Flow Cytometry: A Practical
Approach (second edition, Ormerod MG ed.)
Oxford University Press, New York, NY
6. Krishan A., Cabana R.: 2004. Flow Cytometric
Analysis of Electronic Nuclear Volume and
DNA Content in Normal Mouse Tissue. Cell
Cycle 3:3, 380 - 383.
DNA Prep Reagent Kit Methodology
Cells in Suspension
1. Add 100 µL of single cell suspension or TRBC
to 100 µL LPR to obtain a final cell concentration of 3 - 5 x 106 cells/mL.
2. Vortex and add 2 mL of DNA Prep Stain.
3. Vortex and filter with a 25 µm filter tip into a
sample cup and analyze.
Solid Tissue Specimens
1. Place fresh or frozen tissue in a mincing dish
with 2 mL of PBS and shred completely.
2. Add 100 µL with a final cell concentration of
3 - 5 x 106 cells/mL to 100 µL of LPR and vortex.
3. Add 2 mL of DNA Prep Stain and vortex.
4. Filter with a ~25 µm filter tip into a sample cup
and analyze.
Perform instrument alignment with Flow-Check™
Fluorospheres for the 488 nm laser.
Optimize alignment with DNA Reference
Calibrator (TRBC) for the Mercury Arc Lamp.
Cell Cycle Protocol
Adjust fluorescence channel lower level discriminator
to allow the system to trigger on fluorescent particles.
Adjust the settings to place the TRBC in channel 125.
Adjust the lower level discriminator to partially
eliminate sample debris below the TRBC population.
Establish EV of nuclei at channel 200. Establish
diploid G0/G1 peak of samples at channel 200.
The use of a DNA reference such as TRBC as
an internal standard provides consistent placement
of the diploid peak for the sample being analyzed.
2
CELL CYCLE ANALYSIS
DAPI (FL1)
UV
HG ARC
LAMP
Z488/543 RPC
(DICHROIC)
UG1 (Exciter) 365 nm
560 SP
(SPLITTER)
PMT
FL2
Blocker: 570 LP
PMT
FL1
DAPI
Figure 1. Cell Lab Quanta™ optical configuration with mercury arc lamp configuration.
3
Blocker:
450AF55
CELL CYCLE ANALYSIS
PROPIDIUM IODIDE (FL2)
Laser
488 nm
Z488/543 RPC
(DICHROIC)
460
RBSP
Propidium
Iodide
560 SP
(SPLITTER)
PMT
FL2
Blocker:
570 LP
PMT
FL1
Figure 2. Cell Lab Quanta™ optical configuration with 488 nm diode laser.
4
Blocker: 525
BP
Region Name
DNA
Reference
Calibrator
Color
Counts
9,910
Pct
99.1%
Linear Mean
201.5
Mode
202.0
Half Peak CV
2.17
DNA Reference Calibrator
EV
Figure 3. DNA Reference Calibrator (TRBC) stained with NIM DAPI 10.
Figure 4. CEM Cell Line and DNA Reference Calibrator stained with NIM DAPI 10.
* All trademarks are the property of their respective owners.
B2005-6653
© 2005 Beckman Coulter, Inc.
Printed in the U.S.A. on recycled paper.