Assaying cell cycle status using flow cytometry

Kang Ho Kim

1,*

and Joel M. Sederstrom

Department of Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza,

Houston TX 77030, USA

Cytometry and Cell Sorting Core, Baylor College of Medicine, 1 Baylor Plaza, Houston TX

77030, USA

Abstract

In this unit, we describe two protocols for analyzing cell cycle status using flow cytometry. The

first is based on the simultaneous analysis of proliferation specific marker (Ki-67) and cellular

DNA content, which discriminates resting/quiescent cell populations (G0 cell) and quantifies cell

cycle distribution (G1, S or G2/M, respectively). The second is based on differential staining of

DNA and RNA through co-staining of Hoechst 33342 and Pyronin Y, which is also useful to

identify G0 cells from G1 cells. Along with these methods for analyzing cell cycle status, we

outline the basics of two additional methods for cell proliferation assays and recent updates of

newly-developed fluorophores, which allows multiplex analysis of cell cycle status, cell

proliferation and a gene of interest using flow cytometry.

Keywords

Cell Cycle; Flow Cytometry; Ki-67; Propidium Iodide; Pyronin Y; Hoechst 33342

UNIT INTRODUCTION

Assessing cell cycle distribution and cell proliferation is important for studying cell growth

differentiation, senescence and apoptosis. This enables one to investigate underlying basic

mechanisms as well as to evaluate therapeutic efficacies of anti-cancer drugs. During cell

cycle progression, proliferating cells sequentially undergo a transition of G1→S→G2→M

phases for synthesis of DNA, preparation of cell division and subsequent mitosis process

(Malumbres and Barbacid, 2009). However, under certain circumstances, cells can enter G0

phase, where the cells are neither dividing nor preparing for proliferation. These resting cells

are characterized by having minimal cell cycle machinery and maintaining specialized

Correspondence to; Kang Ho Kim, Ph.D., Department of Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor

Plaza, Houston TX, 77030, USA, Tel: (713) 798-6223, [email protected].

Conflict of Interest

The Authors declare no conflict of interest.

INTERNET RESOURCES

http://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/flow-cytometry/cell-health-and-viability-assays-for-flow-

cytometry/cell-proliferation-assays-for-flow-cytometry/click-it-edu-cell-proliferation-assay-kits-for-flow-cytometry.html

Web site for the introduction of Click-iT® Plus Assay Kits

HHS Public Access

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Published in final edited form as:

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cellular functions rather than by proceeding to cell proliferation. This resting state is also

referred as quiescent (Zetterberg et al., 1995).

The earliest and simplest approach to analyze cell cycle status is to measure cellular DNA

content at a single time point (Darzynkiewicz and Huang, 2004). This reveals a snapshot of

cell cycle status among 3 distinct groups (i.e., G0/G1 (2n), S (2n~4n), and G2/M (4n) phase,

respectively). In 1969, the feulgen-DNA staining method was first described to analyze cell

cycle distribution (Van Dilla et al., 1969). Since then, many fluorescent DNA dyes have

been developed for multiplex analysis of cellular DNA content and other proliferation-

related markers (Table 1). However, this method is insufficient to understand detailed cell

cycle status because DNA content alone cannot distinguish resting/quiescent cells (G0) from

G1 phase cells.

To overcome this limitation, some alternative methods have been developed. First, resting/

quiescent and proliferating cell fractions can be identified by proliferation-associated

proteins and/or nuclear proliferation antigens such as Ki-67 and proliferating cell nuclear

antigen (PCNA). Ki-67 antigen is rarely detected in G0 phase, highly expressed in the

nuclear region of proliferating cells (maximum in G2 and early M phases) and rapidly

degraded during anaphase and telophase of mitosis processes (Gerdes et al., 1984).

Likewise, PCNA is a good marker for proliferating cells and is concentrated in S phase

(Kurki et al., 1986), which is useful to separate S phase cells. Second, quantification of

intracellular RNA by Hoechst 33342/Pyronin Y double staining can be an alternative way to

study cell cycle status because highly proliferating cells usually contain higher levels of

RNA compared to resting/quiescent cells. Historically, Pyronin Y has been widely used for

microscopic observation of cellular RNA in combination with methyl green (Scott, 1967).

Its application was extended to flow cytometry by Howard Shapiro in 1981 and further

defined by Zibgniew Darzynkiewicz in 2004 (Darzynkiewicz et al., 2004; Shapiro, 1981). In

this unit, two basic flow cytometric techniques are described for assessing cell cycle status

through costaining of Ki-67/DNA (Basic Protocol 1) and quantification of intracellular RNA

(Basic Protocol 2).

BASIC PROTOCOL 1

Title

Flow cytometric analysis of Ki-67 and DNA content for analyzing cell cycle status.

Introduction

The Ki-67 antibody was first described to recognize a nuclear protein only present in

proliferating cells (Gerdes et al., 1983). Later, the nuclear antigen detected by Ki-67

antibody was identified as two isoforms of 320 kDa and 359 kDa Ki-67 protein, which may

be necessary for maintenance of cell proliferation (Schluter et al., 1993). The Ki-67-positive

population is mainly limited to proliferating cells in many cell types during active phases of

the cell cycle (G1, S, G2 and M phases), whereas it is absent from resting/quiescent cells

(Gerdes et al., 1984; Schwarting et al., 1986). Thus, Ki-67 has been extensively used to

predict the growth rate of many cancer samples from human patients. This protocol provides

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a detailed procedure for determining cell cycle status of tissue culture cells through double

staining of Ki-67 and PI using flow cytometry.

Materials

Solutions and reagents—1X Phosphate buffered saline (PBS)

70% Cold ethanol (−20°C)

FACS buffer (see recipe)

PI staining solution (see recipe)

FITC-conjugated Ki-67 antibody

NOTE: Alternatively, various fluorescent dyes such as PE and APC can be used for Ki-67 in

combination with other DNA-binding fluorescent dyes (Table 1) in order to avoid

significant spectral overlap.

Special equipment—Flow cytometer equipped with 488 nm blue laser and appropriate

filter sets detecting FITC and PI fluorescence.

Steps and Annotations

Harvest, fix and permeabilize cells

1. Plate cells at proper density so that cells should not be confluent at the time of cell

harvest (See Critical Parameters).

2. Harvest and pellet cells (1 × 10

) after washing with 10 ml PBS by centrifuging 5

min at 200 × g.

3. Remove supernatant and resuspend cells in 0.5 ml PBS

4. Add 4.5 ml pre-chilled 70% cold ethanol (−20°C) in a drop wise manner to the cell

suspension while vortexing.

In this step, cell clumping should be minimized (see Troubleshooting).

5. Incubate the fixed cells at least 2 hour at −20°C. Cells may be stored in ethanol

fixative for several weeks at −20°C prior to antibody staining.

Stain cells with Ki-67 antibody and fluorescent DNA dyes

6. Centrifuge 3 min at 300 × g and remove ethanol.

7. Rinse cells with 5 ml FACS buffer tw1ice by centrifuging 5 min at 200 × g.

8.Remove supernatant and resuspend cells in 100 μl FACS buffer (1 × 10

cells/100 μl).

9. Add 10 μl pre-diluted Ki-67-FITC antibody and incubate 30 min at room

temperature.

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Refer to manufacturer's instruction for optimal antibody dilution. For the best quality of

positive cell discrimination from negative cells, titration of Ki-67-FITC antibody is

required if nothing is specified.

After this step, the rest of the procedure should be performed in the dark.

10. Wash with 5 ml FACS buffer twice by centrifuging 5min at 200 × g.

11. Remove supernatant. Add 500 μl PI staining solution and resuspend pellet gently.

12. Incubate 20 min at room temperature.

Washing is not necessary.

Perform flow cytometry

13. Set up and adjust flow cytometer with a blue laser (488 nm) and detection filters

(530/30 nm band pass for FITC and 610/20 nm band pass for PI).

Ki-67-FITC signal in logarithmic mode and PI signal in linear mode. PI fluorescence

can be detected in 585/42 nm band pass, 670 nm long pass filters or something capable

of detecting PI fluorescence.

14. Set a low flow rate (less than 400 events/second) for optimal resolution of PI

fluorescence.

15. Exclude doublets by creating a combination of same-channel bivariate plots

utilizing Area vs Height or Area vs Width (i.e., FSC, SSC and PI fluorescence).

Singlet events are presented in a diagonal pattern. Doublets have lower Height and

higher Width values.

16. Acquire the fluorescence and analyze cell cycle stages of each sample (See

Anticipated Results).

Appropriate Compensation procedures between fluorophores should be utilized.

ALTERNATE PROTOCOL 1

Title

Simultaneous staining of cell surface antigens with Ki-67 and PI.

Introduction

This method can be used for multiplex staining of surface proteins and Ki-67/DNA, which

enables analysis of surrogate markers for resting/quiescent cells. After first staining of

surface antigen, both fixation and permeabilization are subsequently required. Here, we

describe classical procedures of surface antigen staining followed by fixation (4%

formaldehyde) and permeabilization (1% saponin). The saponin-based permeabilization is a

reversible process so the remainder of the procedure should be performed in a presence of

saponin. This method usually preserves cell shape as well as structure of cellular

components but takes longer time for surface antigen staining, fixation and permeabilization

processes.

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Material List

Solutions and reagents—1X Phosphate buffered saline (PBS)

FACS buffer (see recipe)

Fluorophore-conjugated antibody against surface antigen

Fixation solution (4% paraformaldehyde)

Permeabilization solution (see recipe)

Saponin wash buffer (see recipe)

FITC-conjugated Ki-67 antibody

PI/saponin staining solution (see recipe)

NOTE: Other fixatives and permeabilization buffers are commercially available (FIX &

PERM® Cell Fixation & Cell Permeabiliazation Kit (#GAS003/GAS004) from Life

Technologies; Cytofix/Cytoperm™ Fixation/Permeabiliazation Solution Kit (#554714) from

BD Biosciences, etc). For detailed procedures, refer to manufacturer's instructions.

NOTE: Optimization of paraformaldehyde (1~4%) and saponin (0.1~1%) concentrations are

needed.

Special equipment—Flow cytometer equipped with 488 nm blue laser and appropriate

filter sets for detecting FITC and PI fluorescence. Depending on the fluorophore for surface

antigens, additional laser and filter sets are needed.

Steps and Annotations

Stain cell surface antigen with fluorescent-conjugated antibody

1. Harvest cells (1 × 10

) and wash with 10 ml PBS by centrifuging 5 min at 200 × g.

2. Remove supernatant and resuspend cells in 100 μl FACS buffer.

3. Add non-fixation sensitive fluorophore-conjugated primary antibody detecting cell

surface antigen and incubate 20 min in the dark at 4°C.

The detection wavelengths for the fluorescent dyes for surface antigens should not

overlap with those for FITC and PI. For optimal antibody dilution, refer to the

manufacturer's instructions or perform a titration. After this step, the rest of the

procedure should be done in the dark.

4. Wash with 5 ml FACS buffer twice by centrifuging 5 min at 200 × g.

Fix and permeabilize cells for intracellular staining

5. Resuspend cells in 200 μl Fixation solution (4% paraformaldehyde).

6. Incubate 20 min at room temperature.

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7. Add 5 ml PBS and centrifuge 5 min at 200 × g to remove fixative.

8. Resuspend cells in 200 μl Permeabilization solution and incubate 20 min at room

temperature.

After this step, 0.5% saponin should be present in all buffers used in this protocol.

9. Wash cells with 5 ml Saponin wash buffer and centrifuge 5 min at 200 × g.

Stain with Ki-67 and PI

10. Resuspend cells in 100 μl Saponin wash buffer and add 10 μl pre-diluted Ki-67-

FITC antibody.

Refer to manufacturer's instruction for optimal antibody dilution. For the best quality of

positive cell discrimination from negative cells, titration of Ki-67-FITC antibody is

required

11. Incubate 30 min at room temperature.

12. Wash cells with 5 ml Saponin wash buffer twice by centrifuging 5 min at 200 × g.

13. Add 500 μl PI/saponin staining solution and resuspend pellet gently.

14. Incubate 20 min at room temperature.

Washing is not necessary.

Perform flow cytometry

15. Set up and adjust flow cytometer with proper laser and filter sets

For detecting FITC and PI, a blue laser (488 nm), detection filters of 530/30 nm band

pass (for FITC) and 610/20 nm band pass (for PI) are used. Alternatively, 585/42 nm

band pass, 670 nm long pass filters or something capable of detecting PI fluorescence

can be used. For detecting surface staining, use proper laser and filter sets according to

the selected fluorophore. Ki-67-FITC signal in logarithmic mode and PI signal in linear

mode.

16. Set a low flow rate (less than 400 events/second) for optimal resolution of PI

fluorescence.

17. Exclude doublets by creating a combination of same-channel bivariate plots

utilizing Area vs Height or Area vs Width (i.e., FSC, SSC and PI fluorescence).

Singlet events are presented in a diagonal pattern. Doublets have lower Height and

higher Width values.

18. Acquire the fluorescence and analyze cell cycle stages of each sample.

Appropriate Compensation procedures between fluorophores should be utilized.

BASIC PROTOCOL 2

Title

Pyronin Y and Hoechst 33342 staining for analyzing cell cycle status.

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Introduction

The other way to identify the resting cells (G0 cells) from proliferating cell is to determine

the total RNA content inside the cells. Generally, resting/quiescent cells at G0 phase have

lower levels of RNA compared with proliferating interphase cells (G1-S-G2-M phase). To

address this, double staining of Hoechst 33342 and Pyronin Y is widely used. Pyronin Y

intercalates both double stranded DNA and double stranded RNA, which can be used for

visualization of RNA as an orange-red band during electrophoresis. In the presence of DNA-

chelating fluorescent dye such as Hoechst 33342, interactions of Pyronin Y and DNA

complex are disrupted and Pyronin Y mainly stains RNA (Shapiro, 1981), allowing the

quantification of RNA amount in a single cell level. Here, we describe a basic protocol for

double staining of cells with Pyronin Y and Hoechst 33342 to dissect resting and

proliferating cells.

Material List

Solutions and reagents—1× Phosphate buffered saline (PBS)

70% Cold ethanol (−20°C)

FACS buffer (see recipe)

Hoechst/PY staining solution (see recipe)

Special equipment—Flow cytometer equipped with both 355 nm UV and 488 nm blue

laser to activate Hoechst 33342 and Pyronin Y. 488 nm laser can be replaced by 532 nm

green or 561 nm yellow-green lasers. Appropriate filter sets are needed.

Steps and Annotations

1. Harvest cells (1 × 10

) and wash with 10 ml PBS by centrifuging 5 min at 200 × g.

2. Resuspend cells in 0.5 ml PBS.

3. Fix cells by adding 4.5 ml pre-chilled 70% ethanol (−20°C) drop wise while

vortexing

4. Incubate at least 2 hour at −20°C.

In this step, cell clumping should be minimized (see Troubleshooting). Ethanol-fixed

cells can be stored several weeks at −20°C.

5. Eliminate residual ethanol by centrifuging 3 min at 300 × g. Remove and discard

supernatant.

6. Wash cells with 5 ml FACS buffer twice by centrifuging 5 min at 200 × g.

7. Stain cells using 0.5 ml Hoechst/PY staining solution.

The sample should be kept in the dark. A washing step is not necessary.

8. Incubate 20 min at room temperature and analyze the fluorescences in flow

cytometry.

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Perform flow cytometry

9. Set up and adjust flow cytometer with UV (355 nm) and blue (488 nm) lasers as well

as proper filter sets (450/50 nm band pass for Hoechst and 575/26 nm band pass for

Pyronin Y)

Pyronin Y is excited by 488 nm blue laser and 532 or 561 nm lasers are also available.

Maximal emission wavelength of Hoechst 33342 is 461 nm and that of Pyronin Y is

575 nm. Both Hoechst 33342 and Pyronin Y signal in a linear mode.

10. Exclude doublets by creating a combination of same-channel bivariate plots

utilizing Area vs Height or Area vs Width (i.e., FSC, SSC and Hoechst fluorescence).

Singlet events are presented in a diagonal pattern. Doublets have lower Height and

higher Width values.

11. Acquire the fluorescence and analyze cell cycle stages of each sample (See

Anticipated Results).

Appropriate Compensation procedures between fluorophores should be utilized.

REAGENTS AND SOLUTIONS

FACS buffer

1X PBS supplemented with:

2% (v/v) heat-inactivated, sterile-filtered fetal bovine serum (10 ml FBS per 500 ml)

1 mM EDTA (1 ml of 0.5 M EDTA stock per 500 ml)

Store at 4°C for up to 6 months. 2% (v/v) FBS can be replaced by 0.2~0.5% (w/v) bovine

serum albumin (BSA). Sodium azide (NaN

, 0.1%) can be added to prevent microbial

contamination.

PI staining solution

1X PBS supplemented with:

50 μg/ml propidium iodide (50 μl of 1 mg/ml PI stock per 1 ml)

100 μg/ml RNase (10 μl of 10 mg/ml RNase stock per 1ml)

2 mM MgCl

(2 μl of 1 M MgCl

stock per 1 ml)

Prepare freshly and keep in the dark at 4°C before use.

Permeabilization solution

10 mM HEPES buffer, pH 7.2 (10 μl of 1 M HEPES buffer stock per 1 ml)

1% (w/v) saponin (100 μl of 10% (w/v) saponin stock per 1 ml)

Prepare freshly and store at 4°C before use.

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Saponin wash buffer

10 mM HEPES buffer, pH 7.2 (10 μl of 1 M HEPES buffer stock per 1 ml)

0.5% (w/v) saponin (50 μl of 10% (w/v) saponin stock per 1 ml)

Prepare freshly and store at 4°C before use.

PI/saponin staining solution

Saponin wash buffer (see recipe) supplemented with:

50 μg/ml propidium iodide (50 μl of 1 mg/ml PI stock per 1 ml)

100 μg/ml RNase (10 μl of 10 mg/ml RNase stock per 1ml)

2 mM MgCl

(2 μl of 1 M MgCl

stock per 1 ml)

Prepare freshly and keep in the dark at 4°C before use.

Hoechst/PY staining solution

FACS buffer (see recipe) supplemented with:

2 μg/ml Hoechst 33342 (2 μl of 1 mg/ml Hoechst 33342 stock per 1 ml)

4 μg/ml Pyronin Y (4 μl of 1 mg/ml Pyronin Y stock per 1 ml)

Prepare freshly and keep in the dark at 4°C before use. Pyronin Y concentration may vary

(see Critical Parameters and Troubleshooting)

COMMENTARY

Background Information

Flow cytometric analysis of Ki-67 was described to determine the growth fraction of

lymphoma cell lines (Schwarting et al., 1986) and further applied to a cell cycle and cell

proliferation analysis on many cancer cells and hematopoietic stem cells. Pyronin Y was

first synthesized in 1889 and it has been used as a convenient histological/cytochemical dye

to stain RNA in combination with methyl green (for DNA staining). Later, double staining

of Pyronin Y and Hoechst 33342 was developed for flow cytometric analysis to estimate

DNA and RNA content in intact cells (Shapiro, 1981). These methods have been widely

used for analyzing cell cycle status.

Along with these techniques, the kinetics of cell cycle status can be assessed by cell

proliferation assays based on measuring newly-synthesized DNA content and cellular

metabolism parameters. For flow cytometric analysis of cell proliferation, genomic DNA in

replicating cells can be labeled by exposing cells to thymidine analog, 5'-bromo-2'-

deoxyuridine (BrdU) during the S phase of cell cycle. Incorporated BrdU is further stained

with fluoresceinated anti-BrdU antibodies and fluorescent DNA dye (e.g., propidium iodide,

PI; 7-Aminoactinomycin D, 7-AAD) to separate the cells according to the cell cycle of each

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phase (i.e., G1, S, G2/M phases) (Rothaeusler and Baumgarth, 2007). A disadvantage of

BrdU incorporation method is that both membrane permeabilization and harsh DNA

denaturation processes are required for antibody penetration to the incorporated BrdU. As an

alternative of BrdU, 5-ethynyl-2'-deoxyuridine (EdU) has been developed to overcome the

limitations of BrdU method (Cappella et al., 2008; Cavanagh et al., 2011; Salic and

Mitchison, 2008). After EdU treatment during cell proliferation, incorporation of EdU can

be subsequently detected by a fluorescent azide molecule through a copper (I) catalyzed

reaction which results in a stable triazole ring formation between EdU and fluorescent dye

(called "Click reaction"). Since the small-sized fluorescent dye readily penetrates the cell

and it easily reacts with EdU even in intact DNA double strand, EdU method is highly

sensitive and much faster than a classical BrdU incorporation method. Also, EdU

incorporation assay can be combined with multiplex cell surface/intracellular staining,

which is very useful for many applications (Cappella et al., 2008; Diermeier-Daucher and

Brockhoff, 2010). The original version of the Click reaction cannot be used for multiplex

detection of some fluorophores such as GFP and R-PE which are easily damaged by high

concentration of copper and reactive oxygen species. Recently, chemical modification of

Click reaction enables to preserve GFP and R-PE fluorescence and to obtain a bright EdU

signal. This is extended to cover at least three different fluorophores (Alexa Fluor® 488,

Alexa Fluor® 647 and Pacific Blue™, see the INTERNET RESOURCES below).

Dye dilution assays using membrane-permeable fluorescent dyes are currently used to assess

cell proliferation as well. Carboxyfluorescein succinimidyl ester (CFSE or CFDA-SE,

carboxyfluorescein diacetate succinimidyl ester) is one of the widely-used fluorescent dyes

that enters the cytoplasm and covalently couples to intracellular amino acids (Lyons, 2000;

Lyons et al., 2013). Because this reaction results in extremely long-term retention of

fluorescent dye within the original cell, it was originally used to track immune cells.

Assuming that cells have homogenous cell size and undergo symmetric division, each

daughter cell has half of the parental cell volume and cellular components, as well as labeled

CFSE dyes. Thus, CFSE labeling can be applied to estimate the number of the generation

after rapid cell proliferations. Usually, CFSE dye may be traced through 6-8 generations by

flow cytometry. Similar to CFSE dye, other fluorophores for dye dilution proliferation assay

have been developed for encompassing broad range of excitation/emission spectrum (Table

2). These fluorescent dyes are better suited for multicolor applications where GFP

derivatives or FITC or similar fluorescent-conjugated antibody is used. Further, some of

dilution dyes emit in channels where cells have less natural autofluorescence that can

detected up to 10 generations during cell proliferation.

Critical Parameters

Proper cell density—Cell density should be optimized because confluent cultures may

cause growth arrest by contact inhibition, which leads to G0/G1 arrest of the cell cycle.

Excessive confluency also affects nutrient availability as well as media acidity, which may

distort experimental results. Generally, cells are harvested during the time window of

exponential growth (usually 50~70% confluency).

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Titration of antibody and fluorescent DNA dye concentration—For the first use,

antibodies against Ki-67 or surface antigen need to be titrated (usually 1:500-1:50 for flow

cytometry) to maximize the detection of signal-positive populations. Fluorescent DNA dye

concentrations may vary among cell lines and/or specific conditions, which have to be

determined empirically.

Determination of Pyronin Y concentration—Proper PY concentration is critical. Low

PY concentration does not ensure stoichiometric correlation between actual RNA level and

PY fluorescence. High PY concentration also results in condensation (precipitation) of PY-

nucleic acid complex, which interferes with PY fluorescence. Thus, optimal PY

concentration will vary among cell line, cell density and condition. Generally, titration is

required for the first use (starting from 1 μg/ml to 5 μg/ml).

Fluorescent protein analysis—To utilize BASIC PROTOCOL 1 or 2 in combination

with fluorescent proteins such as GFP and RFP, it is the best to use the ALTERNATIVE

PROTOCOL 1 as the PFA fixation followed by saponin permeabilization helps retain

structure of these proteins. Changes to BASIC PROTOCOL 1 may need to be made as GFP

and FITC spectra overlap. So, consider another non-overlapping probe for detecting Ki-67.

Some RFP derivatives also overlap with PI and, in some cases, alternative DNA probes

should be utilized.

Troubleshooting

Poor positive signal or high background fluorescence—Check appropriate laser

and filter combinations. Adjust concentration and incubation time of antibodies and

fluorescent DNA/RNA dyes. For high background fluorescence, increase FBS/BSA

concentration in the FACS buffer. In some instances (e.g., ALTERNATIVE PROTOCOL

1), PFA concentrations and incubation times may need to be adjusted to reduce background

signals. In cases where the signal is poor or non-existent with regard to surface staining,

check the manufacturer's instructions if the conjugated antibody is fixation sensitive (e.g.,

prolonged exposure to paraformaldehyde affects emission spectra of some fluorophores such

as APC-Cy™7, PE-Cy™7).

Cell clumping and extensive cell loss during fixation/washing process—

Improper fixation procedure may result in cell clumping and significant cell loss. To avoid

this, inject the cell suspension directly into the cold ethanol using a Pasteur pipette and mix

well immediately. Alternatively, use non-alcohol fixatives such as 4% paraformaldehyde

(see ALTERNATIVE PROTOCOL 1). The stained sample should be passed through a cell

strainer before analysis.

High Coefficient of Variation (CV) or wide peaks for DNA cell cycle probes—

Ensure that the samples are run in the lowest sample pressure setting possible to allow for

best interrogation of sample. Acquiring the sample in the linear setting/range of the flow

cytometer is also important. Additionally, proper cell and dye concentration is critical for

consistent histograms giving better CVs and decreasing variation between samples.

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Anticipated Results

On the basis of differences in Ki-67 expression level (Figure 1A) and RNA content (Figure

1B) of G0 cells, Basic Protocol 1 and 2 allow discrimination of resting/quiescent (G0)

population from other proliferating cells (G1, S, G2/M phases). Generally, the G0 cells have

lower levels of Ki-67 and RNA levels, so these cells may be distinguishable from other

proliferating cells. To quantify cell cycle distribution of proliferating cells precisely, fitting

software such as ModFit LT (Verity Software) and MultiCycle AV (Pheonix Flow Systems)

can be used (Darzynkiewicz and Huang, 2004).

Previous studies have demonstrated that a small portion of cells showed a significant

increase of Ki-67 level in G2/M cells (Figure 1A, asterisk) (Landberg et al., 1990). These

cells were regarded as early mitotic cells but this has not yet been firmly established. To

further separate M phase cells, other markers such as Cyclins, MPM-2 and phospho-Ser10-

histone H3 (to detect M phase) need to be combined (Juan and Darzynkiewicz, 2001;

Landberg et al., 1990; Vignon et al., 2013).

Time Considerations

Preparation and staining of Ki-67 and fluorescent DNA dye staining should take 4 hours.

The staining of Pyronin Y and Hoechst 33342 will take 3 hours. Analysis the samples

through flow cytometry will take from 1 to 10 minutes per sample depending on

concentrations. Total time is dependent upon total sample numbers.

Acknowledgement

This work was supported by the NIH (NIDDK R01DK46546) and the Cytometry and Cell Sorting Core at Baylor

College of Medicine with funding from the NIH (NIAID P30AI036211, NCI P30CA125123, and NCRR

S10RR024574)

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Figure 1.

Analysis of cell cycle status by differential staining of Ki-67/DNA and DNA/RNA. HeLa

cells were fixed and subsequently stained with Ki-67-FITC and PI (A) or Hoechst 33342

and Pyronin Y (B) according to the BASIC PROTOCOL 1 and 2.

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Kim and Sederstrom Page 15

Table 1

Lists of common fluorescent DNA dyes

Commercial Name Ex

max

Ex. Laser Manufacturer

DAPI 345 455 UV Various

Hoechst 33342

350 461 UV Various

Propidium Iodide (PI) 535 617

UV, 488, 532 or

561 nm

Various

7-AAD 546 647 488 or 532 nm Various

DRAQ5

647 681/697 633 nm Various

DRAQ7 599/644 694 633 Various

FxCycle

Violet

358 461 405 nm Life Technologies

Vybrant® DyeCycle

Violet

369 437 UV or 405 nm Life Technologies

Vybrant® DyeCycle

Green

506 534 488 nm Life Technologies

Vybrant® DyeCycle

Orange

519 563 488 or 532 nm Life Technologies

Vybrant® DyeCycle

Ruby

638 686 561 or 633 nm Life Technologies

SYTOX® Blue 444 480 405 nm Life Technologies

SYTOX® Green 504 523 488 nm Life Technologies

SYTOX® Orange 547 570 488 or 532 nm Life Technologies

SYTOX® AADvanced TM 546 647 488 or 532 nm Life Technologies

SYTOX® Red 640 658 633 nm Life Technologies

FxCycle

Far Red

640 658 633 nm Life Technologies

cell permeable dye which can stain DNA in both live or fixed cells.

Curr Protoc Mol Biol. Author manuscript; available in PMC 2016 July 01.

Author Manuscript Author Manuscript Author Manuscript Author Manuscript

Kim and Sederstrom Page 16

Table 2

Lists of fluorophores for dye dilution proliferation assay

Commercial Name Ex

max

Ex. Laser Manufacturer

CFSE 495 519 488 nm Various

CellTrace

Violet

405 450 405 nm Life Technologies

BD HorizonTM Violet Cell

Proliferation Dye (VPD450)

404 448 405 nm BD Biosciences

Cell Proliferation Dye eFluor®450 405 450 405 nm eBioscience

CytoTrack

Blue

403 454 405 nm BIO-RAD

Oregon Green 488 carboxylic acid

diacetate, SE (carboxy-DFFDA SE)

496 524 488 nm Life Technologies

SNARF-1 carboxylic acid, acetate, SE 514 586 488 nm Life Technologies

CytoTrack

Green

511 525 488 nm BIO-RAD

CytoTrack

Yellow

542 556 532 nm BIO-RAD

CellTrace

Far Red DDAO-SE

647 657 633 nm Life Technologies

Cell Proliferation Dye eFluor®670 647 670 633 nm eBioscience

CytoTrack

Red

628 643 633 nm or 640 nm BIO-RAD

Curr Protoc Mol Biol. Author manuscript; available in PMC 2016 July 01.