q 2006 International Society for Analytical Cytology
Cytometry Part A 69A:1028–1036 (2006)
Factors Affecting Flow Karyotype Resolution
Bee Ling Ng* and Nigel P. Carter
The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom
Received 20 February 2006; Revision Received 17 May 2006; Accepted 19 June 2006
Background: One of the major factors which influences
the chromosome purity achievable particularly during high
speed sorting is the analytical resolution of individual chromosome peaks in the flow karyotype, as well as the amount
of debris and fragmented chromosomes. We have investigated the factors involved in the preparation of chromosome suspensions that influence karyotype resolution.
Methods: Chromosomes were isolated from various
human and animal cell types using a series of polyamine
buffer isolation protocols modified with respect to pH,
salt concentration, and chromosome staining time. Each
preparation was analyzed on a MoFlo sorter (DAKO) configured for high speed sorting and the resolution of the
flow karyotypes compared.
Results: High resolution flow cytometric data was obtained with chromosomes optimally isolated using hypotonic solution buffered at pH 8.0 and polyamine isolation
buffer (with NaCl excluded) between pH 7.50 and 8.0.
High purity chromosomes are required for making FISH
paints (1–9), creating specific DNA libraries (1–7), and for
applications using microarrays (8–15). While contributing
factors from the fluidics and optics of the flow system
clearly influence the resolution of chromosomes analysis
and so the purity at which chromosomes can be isolated
by flow sorting, equally important is the chromosome
preparation itself. It has been well established that the resolution of individual chromosomes in the flow karyotype,
as well as the amount of small DNA debris and the presence of fragmented and clumped chromosomes, is influenced by the presence of stabilizing agents (e.g. polyamines, magnesium ions) (16–18), concentration of NaCl
(17), as well as the pH (18) used in the isolation buffers.
Several chromosome isolation methods have been used in
the study of parameters that influence the resolution of
the flow karyotype and factors such as pH (18), ion composition of buffer (17,18), mitotic index (19,20), and
kinetics of DNA staining (21) have been studied mainly for
univariate analysis. These parameters contribute to how
well the chromosomes are resolved from each other, the
frequency of chromosome clumps, and the amount of
DNA-containing debris (16–18,22–24).
In this paper, we investigate the factors that affect the
resolution of the bivariate flow karyotype from chromo-
Extending staining time to more than 8 h with chromosome suspensions isolated from cell lines subjected to sufficient metaphase arrest times gave the best result with
the lowest percentage of debris generated, tighter chromosome peaks with overall lower coefficients of variation,
and a 1- to 5-fold increase in the yield of isolated chromosomes.
Conclusions: Optimization of buffer pH and the length
of staining improved karyotype resolution particularly for
larger chromosomes and reduced the presence of chromosome fragments (debris). However, the most interesting
and surprising finding was that the exclusion of NaCl in
PAB buffer improved the yield and resolution of larger chromosomes. q 2006 International Society for Analytical Cytology
Key terms: chromosomes; metaphase; flow karyotype;
resolution; flow cytometry; high speed sorting; purity
somes prepared from various types of human and animal
cell lines using a polyamine buffer.
MATERIALS AND METHODS
Cell Culture
Chromosomes were prepared from various cells: (1)
human lymphoblastoid cells GM11321B (derived from
normal human male blood), 1183t(2;17), DD3606t(3;11);
(2) lipopolysaccaride stimulated B lymphocytes from the
c57/BL6 mouse strain; (3) armadillo fibroblast cell line.
Lymphoblastoid and fibroblast cell lines were cultured in
RPMI medium (Gibco) and DMEM (Gibco) medium respectively and supplemented with 15% fetal bovine serum (FBS,
Gibco) and antibiotics (Penicillin and Streptomycin, Sigma).
Near confluent cells were subcultured to 50% 24 h before
treatment with demecolcine (0.1 lg /ml).
B lymphocytes from the spleen of a c57/BL6 mouse
(male) were prepared and stimulated using lipopolysacharide (LPS, Sigma) as described previously (25). After
*Correspondence to: Bee Ling Ng.
E-mail: [email protected]
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI: 10.1002/cyto.a.20330
FLOW KARYOTYPE
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FIG. 1. Bivariate flow karyotypes of chromosomes from a normal male human lymphoblastoid cell line, GM11321B. The flow karyograms are displayed as density
plots. (i) Hypotonic solution kept constant at pH 6.60 (unadjusted value). The pH value of PAB buffer was varied from 6.0 to 9.0. (ii) PAB buffer kept constant at pH
7.0 (unadjusted value). The pH value of hypotonic solution was varied from 6.0 to 9.0. The percentage of debris (de) generated during the chromosome isolation process was calculated by drawing a quadrant on the debris region and is shown in each panel. DNA containing debris is located at the lower left hand corner of the plots.
48 h of culture in LPS (50 lg/ml), the stimulated culture
was blocked at metaphase with 0.1 lg/ml demecolcine
for between 3 and 4 h prior to harvesting.
Chromosome Preparation
Chromosomes were prepared by using modifications of
a polyamine isolation method (16).
For lymphoblastiod cell lines and stimulated B cells,
50 ml of blocked cell culture was centrifuged at 289g for
5 min and the cell pellet resuspended in 5 ml of hypotonic
solution (75 mM KCl, 10 mM MgSO4, 0.2 mM spermine,
0.5 mM spermidine, pH 8.0 unless detailed otherwise)
and incubated at room temperature for 10 min.
For adherent cell lines, the supernatant from eight flasks
(150 cm2) after mitotic shake-off were harvested and centrifuged at 289g for 5 min. The cell suspensions were
pooled together into one tube after resuspending the cell
pellets in hypotonic solution (5 ml).
The suspension of swollen cells was centrifuged at 289g for
5 min. The cell pellet was resuspended in 3 ml of ice-cold polyamine isolation buffer (PAB, containing 15 mM Tris, 2 mM
EDTA, 0.5 mM EGTA, 80 mM KCl, 3 mM dithiothreitol, 0.25%
Triton X-100, 0.2 mM spermine, 0.5 mM spermidine, 20 mM
NaCl, pH 7.50 unless detailed otherwise) and vortexed for 20 s.
20-lm mesh filter (Celltrics, Partec) before staining overnight (unless detailed otherwise), with 5 lg/ml of Hoechst
(Sigma), 40 lg/ml of Chromomycin A3 (Sigma), and
10 mM MgSO4. About 10 mM of sodium citrate and
25 mM of sodium sulphite was added to the stained preparation 1 h before flow analysis.
Effect of pH. The effect of the pH of the hypotonic solution and of the polyamine buffer on flow karyotype resolution was investigated using the human lymphoblastoid
cell line (GM11321B). In one series of experiments, the pH
of the hypotonic solution was maintained at 6.60 (unadjusted value), and the pH of the PAB buffer was varied from
6.0 to 9.0. In a second set of experiments, the pH of the
PAB buffer was kept constant at 7.0, and the pH for the hypotonic solution was varied from 6.0 to 9.0. The optimal
pH ranges obtained from these two experiments were then
refined using a combination of pH values at 0.5 increments.
Effect of NaCl concentration. The concentration of
NaCl in the PAB buffer was varied from 0 to 100 mM for chromosomes prepared from a human lymphoblastoid cell line
(1183t(2;17)) and an animal fibroblast cell line (Armadillo).
Effect of staining time. Chromosomes were stained
with Hoechst 33258 (HO) and Chromomycin A3 (CA3) for
varied times between 1 and 8 h.
Chromosome Staining
Flow Cytometric Analysis
All chromosome suspensions were briefly centrifuged
(201g, 2 min) and the supernatant was filtered through a
Stained chromosome suspensions were analyzed on a
flow cytometer (MoFloÒ, DAKO) equipped with two water-
Cytometry Part A DOI 10.1002/cyto.a
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Table 1
Coefficient of Variation and, Percentage of Gated Events of Chromosome 1 and 18 for Chromosome Preparationsa
from a Normal Male Human Lymphoblastoid Cell Line, GM11321B
Hypotonic pH 6.60
Chromosome 1
Coefficient
variation
PAB pH
HO
CA3
Percentage of
gated events
(fold increase
over pH 6.0)
6.0
7.0
8.0
9.0
1.81
1.19
1.43
1.61
1.88
2.31
2.09
1.72
0.48
1.07 (12.2x)
1.13 (12.4x)
0.32 (20.7x)
Chromosome 18
Coefficient
variation
HO
CA3
Percentage of
gated events
(fold increase
over pH 6.0)
3.06
2.52
2.55
2.92
3.12
3.12
3.74
3.07
0.90
2.26 (12.5x)
2.40 (12.7x)
1.87 (12.1x)
a
Chromosomes are prepared using hypotonic solution with the pH maintained at 6.60 and PAB buffer with the pH varied from 6.0 to 9.0.
cooled lasers (Coherent, Innova 300 series) spatially separated at the flow chamber. The first laser was tuned to emit
multiline UV (330–360 nm) which efficiently excites
Hoechst. The second laser was tuned to emit light at 457
nm to excite CA3. The power of both lasers was set to 300
mW and kept constant using light control feedback. Fluorescence emitted from HO was collected using a 400 nm
long pass filter combined with a 480 nm short pass filter.
Chromomycin fluorescence was collected using a 490 nm
long pass filter. The instrument was also configured for
high speed sorting at a data rate of 8,000–12,000 events
per second with optimal setting of the sheath pressure to
60 psi and the drop drive frequency to 95 kHz using a
70 lm cytonozzle tip.
The optical light path of the flow cytometer was aligned
before chromosome analysis using 3 lm beads (Spheroä
Rainbow Fluorescent particles, Spherotech) with minimum peak coefficient of variance (CV) for both fluorescence channels.
Data for forward scatter, HO fluorescence, and Chromomycin fluorescence were collected using HO fluorescence as
the trigger signal. Flow karyotypes for all cell lines were displayed as a bivariate flow karyogram of HO versus Chromomycin fluorescence after gating on low forward scatter and
high HO fluorescence to exclude some debris and clumps. A
total of 50,000–100,000 events were acquired for each cell
line at a rate of 1,000 events per second. Data collected from
the experiments were analyzed using Summit V3.1 (analysis
software from DAKO) and WinMDI V2.8 (http://www.cyto.
purdue.edu/flowcyt/software/Winmdi.htm). For the calculation of CV, ellipsoid regions were placed around the chromosomes of interest on the bivariate flow karyogram and the
half-max CVs of the gated regions were calculated univariately for HO and Chromomycin fluorescence using the standard feature of the Summit V3.1 program.
RESULTS
The Effect of Buffer pH on the Isolated
Chromosomes
The effect of PAB pH when the pH of the hypotonic solution was maintained at 6.60 is shown in Figures 1A–1D
and Table 1. The quantity of debris generated was reduced
by more than 60% when the pH of the PAB was increased
from 6.0 to 8.0. The lowest amount of debris was measured between pH 7.0 (30%) and 8.0 (25%). The yield of
chromosomes (percentage of gated events) was assessed
and the CV for HO and CA3 fluorescence were measured
for chromosomes 1 and 18 (Table 1). The optimum yields
were found at PAB pH 8.0 and represented 2.4- and 2.7fold increases for chromosomes 1 and 18 respectively over
the yields obtained at PAB pH 6.0. The lowest CVs for HO
fluorescence were found between PAB pH 7.0 and 8.0.
However, for CA3 fluorescence, no significant improvements in the CVs were observed.
In the second series of experiments, the effect of hypotonic pH when the pH of the PAB buffer was maintained
at 7.0 is shown in Figures 1E–1H and Table 2. The amount
Table 2
Coefficient of Variation and Percentage of Gated Events of Chromosome 1 and 18 for Chromosome Preparationsa
from a Normal Male Human Lymphoblastoid Cell Line, GM11321B
PAB pH 7.0
Chromosome 1
Coefficient
variation
Hypotonic pH
HO
CA3
Percentage of
gated events
(fold increase
over pH 6.0)
6.0
7.0
8.0
9.0
1.38
1.40
1.35
2.08
2.12
1.91
1.87
2.12
0.89
1.01 (11.1x)
1.31 (11.5x)
1.21 (11.4x)
Chromosome 18
Coefficient
variation
HO
CA3
Percentage of
gated events
(fold increase
over pH 6.0)
1.95
2.02
1.97
2.86
3.85
3.07
3.12
3.16
1.75
1.98 (11.1x)
2.82 (11.6x)
2.68 (11.5x)
a
Chromosomes prepared using hypotonic solution with the pH varied from 6.0 to 9.0 and PAB buffer with the pH maintained at 7.0.
Cytometry Part A DOI 10.1002/cyto.a
FLOW KARYOTYPE
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FIG. 2. Bivariate flow karyotype of chromosomes from a normal male human lymphoblastoid cell line, GM11321B. Chromosomes are prepared using hypotonic solution and PAB buffer using a combination of pH values at 0.5 increments from pH 7.0 to 8.0. All the isolated chromosomes are stained overnight
with HO and chromomycin before analysis. The flow karyograms are displayed in density plot. The percentage of debris (de) generated during the chromosome isolation process was calculated by drawing a quadrant on the debris region and is shown in each panel. DNA containing debris is located at the lower
left hand corner of the plots.
of debris generated was reduced to more than 40% when
the pH of hypotonic solution was increased from 6.0 to
8.0. The lowest amount of debris was measured at pH 8.0
(21%). The yield of chromosomes (percentage of gated
events) was assessed and the CVs for HO and CA3 fluorescence were measured for chromosomes 1 and 18
(Table 2). The optimum yield of chromosomes was found
at hypotonic pH 8.0 and represented 1.5- and 1.6-fold
increases for chromosomes 1 and 18 respectively over the
yields obtained at hypotonic pH 6.0. The lowest CVs for
CA3 fluorescence were found between hypotonic pH 7.0
and 8.0. However, for HO fluorescence, no significant
improvements in the CVs were observed.
The optimal pH range of 7.0–8.0 obtained from the
above experiments was then refined using a combination
of pH values at 0.5 increments. The data obtained from
this experiment is shown in Figure 2 and Table 3. The lowest amount of debris (15%) was measured when hypo-
Table 3
Percentage of Gated Events of Chromosome 1 and 18 for Chromosome Preparationsa from GM11321B Lymphoblastoid Cell Culture
Hypotonic pH
7.0
7.50
8.0
PAB pH 7.0
Chromo 1
Chromo 18
0.22
0.91
0.97
a
0.46
1.66
1.95
PAB pH 7.50
Chromo 1
Chromo 18
1.44
1.47 (11.0x)
1.69 (11.2x)
2.24
2.63 (11.2x)
2.86 (11.3x)
PAB pH 8.0
Chromo 1
Chromo 18
1.19
1.31 (11.1x)
1.67 (11.4x)
2.25
2.32 (11.0x)
2.87 (11.3x)
Chromosomes are prepared using hypotonic solution and PAB buffer using a combination of pH values at 0.5 increments from pH 7.0
to 8.0. Percentage (%) of gated events (fold increase over hypotonic pH 7.0).
Cytometry Part A DOI 10.1002/cyto.a
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FIG. 3. Bivariate flow karyotype of chromosomes from human lymphoblastoid cell line (i) 1183t(2;17) and an animal fibroblast cell line (ii) Armadillo.
The chromosomes are prepared using polyamine isolation method with the concentration of NaCl in the PAB buffer varied from 0 to 100 mM. All chromosomes are displayed in A, C, and D panel. Panel (B) shows the zoom-in data gated on the first four chromosomes of the cell line. For Armadillo cell line, the
zoom in data is displayed inset the data plot.
tonic solution and PAB buffer were buffered at pH 8.0 and
pH 7.50, respectively. The optimum yield of chromosomes
was found at hypotonic pH 8.0 and between PAB pH
7.50–8.0 (Table 3), and represented 1.3-fold increases for
chromosomes 1 and 18 over the yields obtained at hypotonic pH 7.0.
Table 4
Coefficient of Variation and Percentage of Gated Events of derivative 2, Chromosome 1 Homolog, and Chromosome 18
for Chromosome Preparationsa from 1183t(2;17) Lymphoblastoid Cell Culture
der 2
Percentage (%)
of gated events
(fold increase over
100 mM NaCl)
CA3
Coefficient
variation
[NaCl]n in PAB
Ho
0
10
20
50
75
100
1.04
1.04
1.23
1.05
0.88
0.71
1.84
1.69
2.04
1.67
2.06
1.49
0.94 (11.7x)
0.78 (11.4x)
0.57 (11.0x)
0.55 (20.9x)
0.58 (11.0x)
0.56
Homolog 1a
Coefficient
Percentage (%)
variation
of gated events
(fold increase over
100 mM NaCl)
Ho
CA3
1.31
1.12
1.69
1.33
1.14
1.33
2.05
2.11
2.07
1.33
1.73
2.50
1.02 (11.6x)
0.94 (11.5x)
0.66 (11.0x)
0.65 (11.0x)
0.74 (11.2x)
0.64
Chromosome 18
Coefficient
Percentage (%)
variation
of gated events
(fold increase over
Ho
CA3
100 mM NaCl)
2.23
2.68
3.12
2.68
2.23
2.21
3.53
3.48
4.58
3.95
3.39
3.85
2.65 (11.1x)
2.41 (21.0x)
2.08 (20.8x)
2.33 (20.9x)
2.27 (20.9x)
2.49
a
Chromosomes are prepared using a polyamine isolation method with the concentration of NaCl in the PAB buffer varied from 0 mM
to 100 mM.
Cytometry Part A DOI 10.1002/cyto.a
FLOW KARYOTYPE
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FIG. 4. Bivariate flow karyotype of chromosomes from human lymphoblastoid cell lines (i) DD3606t(3;11) and (ii) c57/BL6 LPS stimulated B lymphocyte
mouse cell culture. Chromosomes were stained with 5 lg/ml of HO 33258 and 40 lg/ml of CA3 for varied times between 1 and 8 h before being analyzed
on a flow cytometer. The zoom in data for DD3606 (9–12 cluster) and LPS stimulated lymphocyte culture is displayed inset the data plot.
The Influence of NaCl on the Resolution
of the Flow Karyotype
The resolution of the flow karyotype was observed to
deteriorate as the concentration of NaCl increased in the
PAB buffer. This reduction in resolution was observed in
particular to affect the large chromosomes such as chromosomes 1 and 2 (see Fig. 3B). The chromosome cluster labeled as derivative 2 of 1183t(2;17) cell line was clearly
resolved from the Chromosome 1 homolog in the absence
of NaCl in the PAB buffer (Fig. 3B). In contrast, the presence
of NaCl in the PAB buffer decreased the resolution of this
chromosome cluster. The yield of chromosomes (percentCytometry Part A DOI 10.1002/cyto.a
age of gated events) was assessed and the CVs for HO and
CA3 fluorescence were measured for derivative 2, Chromosome 1a, and 18 (Table 4). The optimum yields of chromosomes were found in the absence of NaCl (0 mM) in PAB
buffer and represented 1.7-, 1.6-, and 1.1-fold increases for
derivative 2, Chromosome 1a, and 18 respectively over the
yields obtained at 100 mM NaCl in PAB. However, no significant improvements in the CVs were observed.
In a similar experiment, the affect of NaCl in the PAB
buffer on an animal cell line from the armadillo is shown
in Figures 3C and 3D. At 50 mM NaCl in PAB buffer, almost
all the larger set of chromosomes are lost (Fig. 3D). How-
NG AND CARTER
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Table 5
Coefficient of Variation of Various Chromosome Peaks for Chromosome Preparations from DD3606t(3;11) Lymphoblastoid Cell Line
Stained with Hoechst 33258 (HO) and Chromomycin A3 (CA3) for Varied Times Between 1 and 8 h
1 h staining time
Coefficient of variation
Chromosomes
type
1
2
4
X
18
der 3
7 h staining time
Coefficient of variation
Hoechst
CA3
Hoechst
(% over 1 h staining time)
CA3
(% over 1 h staining time)
1.51
1.07
1.19
1.74
3.22
1.24
2.43
1.97
2.68
2.42
3.95
2.01
1.69 (111.9%)
1.25 (116.8%)
1.19 (0%)
1.51 (213.2%)
2.76 (214.3%)
1.25 (10.8%)
1.69 (230.5%)
1.79 (29.1%)
2.40 (210.4%)
2.12 (212.4%)
3.30 (216.5%)
1.54 (223.4%)
ever, a well resolved chromosome profile was obtained in
the absence of NaCl in PAB buffer (Fig. 3C).
The Effect of Staining Time
19 respectively over the CVs obtained at 2 h staining time.
The lowest CVs for CA3 fluorescence were found at 8 h
staining time and represented between 44.8% and 55.9%
improvements for Chromosome 1, 2, X, 3, Y, and 19 respectively over the CVs obtained at 2 h staining time.
The effect of staining time on the apparent resolution
of flow karyotype of a human lymphoblastoid cell line
(DD3606t(3;11)) is shown in Figures 4A and 4B and Table
5. The best apparent resolution of the flow karyotype was
observed when the chromosomes were stained for 7 h on
ice. This can be seen in the detailed view of the human
chromosome 9–12 cluster (inset Fig. 4A and 4B). This
chromosome group, normally a single cluster, showed
improved structure with multiple individual clusters after
7 h staining time (Fig. 4B). The CVs for HO and CA3 fluorescence were measured for chromosomes 1, 2, 4, X, 18,
and derivative 3 (Table 5). The lowest CVs for CA3 fluorescence were found at 7 h staining time and represented
30.5%, 9.1%, 10.4%, 12.4%, 16.5%, and 23.4% improvement for Chromosome 1, 2, 4, X, 18, and derivative 3
respectively over the CVs obtained at 1 h staining time.
However, for HO fluorescence, most of the chromosomes
measured did not show an improved CV except for Chromosome X (13.2%) and 18 (14.3%).
In another experiment, the effect of staining time on LPS
stimulated mouse B lymphocytes culture was investigated
(Figs. 4C and 4D and Table 6). The CVs for HO and CA3 fluorescence were measured for Chromosomes 1, 2, X, 3, Y,
and 19 (Table 6). The lowest CVs for HO fluorescence were
found at 8 h staining time and represented between 2.0%
and 16.8% improvements for chromosome 1, 2, X, 3, Y, and
DISCUSSION AND CONCLUSIONS
Various chromosome isolation procedures (6,16,18,22–
24,26–29) have been developed and adapted for generation of
flow karyotypes. The factors influencing the quality of chromosome preparations using chromosome stabilization based on
magnesium ions have been investigated previously (18,22,23).
We typically use chromosome stabilization buffers based on
polyamines as this approach generates the high molecular
weight DNA required for DNA library construction and other
applications. To obtain high resolution flow karyotype as well
as to isolate chromosomes rapidly, several modifications were
made to the polyamine buffer method used to prepare chromosome suspension. The resolution of the flow karyotype
was assessed based on the HO and CA3 CVs of selected chromosome peaks. The quality of the chromosome preparation
was determined based on the amount of debris generated. The
optimal condition used for the isolation of chromosomes using
polyamine buffer is summarized in Table 7.
The pH of the buffers used in the preparation of chromosome suspensions is an important factor. Previous studies (18,27,28,30) have shown the influence of pH on the
stability as well as on the physical structure of chromosomes. We found that when the pH of the PAB buffer was
between pH 7.0 and 8.0, tighter chromosome peaks were
produced with the effect restricted to a reduction of the
Table 6
Coefficient of Variation of Various Chromosome Peaks for Chromosome Preparations from LPS Stimulated Mouse Lymphocyte
Culture Stained with Hoechst 33258 (HO) and Chromomycin A3 (CA3) for Varied Times Between 1 and 8 h
2 h staining time
Coefficient of variation
8 h staining time
Coefficient of variation
Chromosomes
type
Hoechst
CA3
Hoechst
(% over 2 h staining time)
CA3
(% over 2 h staining time)
1
2
X
3
Y
19
1.49
1.61
1.63
1.58
2.41
2.97
4.31
3.94
4.68
3.88
5.36
5.58
1.46 (22.0%)
1.57 (22.5%)
1.42 (212.9%)
1.35 (214.6%)
2.37 (21.7%)
2.47 (216.8%)
1.90 (255.9%)
1.96 (250.3%)
2.07 (255.8%)
2.09 (246.1%)
2.96 (244.8%)
2.65 (252.5%)
Cytometry Part A DOI 10.1002/cyto.a
FLOW KARYOTYPE
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Table 7
Optimal Procedure for Preparation of Chromosomes Using Polyamine Buffer
1. Arrest cells at metaphase using 0.1 lg/ml demecolcine for optimal amount of time, dependent on the cell cycle time of the cell lines.
(Approximately 5 h for suspension, 16 h for adherent cell lines and 4 h for LPS stimulated B lymphocyte culture).
2. Harvest cells and centrifuge at 289g for 5 min. Remove supernatant.
3. Resuspend cell pellet in 5 ml of hypotonic solution (75 mM KCl, 10 mM MgSO4, 0.2 mM spermine, 0.5 mM spermidine, pH 8.0) and
incubate at room temperature for 10 min.
4. Centrifuge cell suspension at 289g for 5 min. Remove supernatant.
5. Resuspend cell pellet in 3 ml of ice cold polyamine isolation buffer (PAB, containing 15 mM Tris, 2 mM EDTA, 0.5 mM EGTA, 80 mM
KCl 3 mM dithiothreitol, 0.25% Triton X-100, 0.2 mM spermine, 0.5 mM spermidine, pH 7.50) and vortex for 20 s.
6. Briefly centrifuge chromosome suspensions at 201g for 2 min. Filter supernatant through 20 lm mesh filter.
7. Stain chromosomes overnight with 5 lg/ml of Hoechst, 40 lg/ml chromomycin A3 and 10 mM MgSO4.
8. To the stained chromosome suspension, add 10 mM of sodium citrate and 25 mM of sodium sulphite 1 h before flow analysis.
CVs in HO fluorescence. Conversely, when the pH of the
hypotonic solution was between pH 7.0 and 8.0, tighter
chromosome peaks were also produced but with the
effect being restricted to a reduction of the CVs for CA3
fluorescence. We also found the optimal chromosome
yield was achieved when the hypotonic solution was pH
8.0 and the PAB was between pH 7.5 and 8.0.
Degradation of chromosomes can occur during the chromosome isolation process (20). This often results in the
under-representation of the larger chromosomes. It has
been reported that with DNA in solution, the concentration
of NaCl affects the solubilization state of DNA in the presence of polyamines (31). We found an increase in chromosome yield and a decrease in debris when NaCl is excluded
from the PAB buffer (Fig. 3 and Table 4). The counterioninduced condensation of DNA influenced by polyamines
and monovalent cations (31–37) could well influence the
loss of the larger chromosomes perhaps because of changes
in resistance to shearing during vortexing or syringing.
The isolation of large numbers of intact metaphase
chromosomes can only be achieved when the cell culture
is adequately arrested at metaphase. Poor resolution flow
karyotypes often occur with a low mitotic index as a consequent of insufficient metaphase arresting time. Interphase cells are known to be the contributing source for
debris formation (38) during the isolation process. A well
resolved flow karyotype from a chromosome suspension
containing large numbers of isolated intact chromosomes
and low quantity of DNA containing debris was obtainable
for most lymphoblastoid cell lines (data not shown) after
5 h incubation with demecolcine. Chromosome suspensions containing a high concentration of intact single
chromosomes and low amounts of debris are vital for high
speed chromosome sorting (39).
The degree to which the chromosomes are resolved
from each other is very much dependent on the kinetics
and interaction of the fluorochromes with the DNA
(21,40–42). For optimum staining, sufficient time is
required to allow the fluorochromes to equilibrate with
the DNA. We found that extending the staining time
beyond the 2 h we have used previously improved the resolution of the flow karyotype. Chromosomes normally
seen as a single-multi-chromosome cluster (such as human
Chromosomes 9–12 and many mouse chromosomes)
became more clearly resolved into multiple clusters after
an extended staining time of 7–8 h.
Cytometry Part A DOI 10.1002/cyto.a
We were able to generate high resolution flow karyotypes with low quantities of debris from human as well as
animal cell lines from chromosome suspensions prepared
using the optimal conditions (Table 7). We were able to
flow sort at 8,000–12,000 chromosomes per second
without loss of resolution using a conventional high speed
sorter (MoFloÒ, DAKO) using such chromosome preparations thus facilitating the sorting of chromosomes for
library construction and other applications.
ACKNOWLEDGMENTS
We thank Dr. Fengtang Yang, who kindly provided the
armadillo cell line, and Dr. Ruby Banerjee, for help with
preparation of mouse spleens.
LITERATURE CITED
1. Fuscoe JC, McNinch JS, Collins CC, Van Dilla MA. Human chromosome-specific DNA libraries: Construction and purity analysis. Cytogenet Cell Genet 1989;50:211–215.
2. Deaven LL, Van Dilla MA, Bartholdi MF, Carrano AV, Cram LS, Fuscoe
JC, Gray JW, Hildebrand CE, Moyzis RK, Perlman J. Construction of
human chromosome-specific DNA libraries from flow-sorted chromosomes. Cold Spring Harb Symp Quant Biol 1986;51 (Part 1):159–167.
3. Fuscoe JC, Clark LM, Van Dilla MA. Construction of fifteen human
chromosome-specific DNA libraries from flow-purified chromosomes.
Cytogenet Cell Genet 1986;43(1/2):79–86.
4. Fuscoe JC. Human chromosome-specific DNA libraries: Use of an
oligodeoxynucleotide probe to detect non-recombinants. Gene 1987;
52(2/3):291–296.
5. Van Dilla MA, Deaven LL. Construction of gene libraries for each
human chromosome. Cytometry 1990;11:208–218.
6. Lalande M, Kunkel LM, Flint A, Latt SA. Development and use of metaphase chromosome flow-sorting methodology to obtain recombinant
phage libraries enriched for parts of the human X chromosome. Cytometry 1984;5:101–107.
7. Fantes JA, Green DK, Sharkey A. Chromosome sorting by flow cytometry. Production of DNA libraries and gene mapping. Methods Mol
Biol 1994;29:205–219.
8. Gribble SM, Prigmore E, Burford DC, Porter KM, Ng BL, Douglas EJ,
Fiegler H, Carr P, Kalaitzopoulos D, Clegg S, Sandstrom R, Temple IK,
Youings SA, Thomas NS, Dennis NR, Jacobs PA, Crolla JA, Carter NP.
The complex nature of constitutional de novo apparently balanced
translocations in patients presenting with abnormal phenotypes.
J Med Genet 2005;42:8–16.
9. Fiegler H, Gribble SM, Burford DC, Carr P, Prigmore E, Porter KM,
Clegg S, Crolla JA, Dennis NR, Jacobs P, Carter NP. Array painting: A
method for the rapid analysis of aberrant chromosomes using DNA
microarrays. J Med Genet 2003;40:664–670.
10. Gribble S, Ng BL, Prigmore E, Burford DC, Carter NP. Chromosome paints
from single copies of chromosomes. Chromosome Res 2004;12:143–151.
11. Gribble SM, Fiegler H, Burford DC, Prigmore E, Yang F, Carr P, Ng BL,
Sun T, Kamberov ES, Makarov VL, Langmore JP, Carter NP. Applications of combined DNA microarray and chromosome sorting technologies. Chromosome Res 2004;12:35–43.
12. Woodfine K, Fiegler H, Beare DM, Collins JE, McCann OT, Young BD,
Debernardi S, Mott R, Dunham I, Carter NP. Replication timing of the
human genome. Hum Mol Genet 2004;13:191–202.
1036
NG AND CARTER
13. Fauth C, Gribble SM, Porter KM, Codina-Pascual M, Ng BL, Kraus J,
Uhrig S, Leifheit J, Haaf T, Fiegler H, Carter NP, Speicher MR. Microarray analyses decipher exceptional complex familial chromosomal
rearrangement. Hum Genet 2006:1–9.
14. Fiegler H, Carter NP. Genomic array technology. Methods Cell Biol
2004;75:769–785.
15. Woodfine K, Carter NP, Dunham I, Fiegler H. Investigating chromosome organization with genomic microarrays. Chromosome Res 2005;
13:249–257.
16. Sillar R, Young BD. A new method for the preparation of metaphase
chromosomes for flow analysis. J Histochem Cytochem 1981;29:74–
78.
17. Bijman JT. Optimization of mammalian chromosome suspension preparations employed in a flow cytometric analysis. Cytometry 1983;3:
354–358.
18. van den Engh G, Trask B, Cram S, Bartholdi M. Preparation of
chromosome suspensions for flow cytometry. Cytometry 1984;5:
108–117.
19. Matsson P, Rydberg B. Analysis of chromosomes from human peripheral lymphocytes by flow cytometry. Cytometry 1981;1:369–372.
20. Young BD, Ferguson-Smith MA, Sillar R, Boyd E. High-resolution analysis of human peripheral lymphocyte chromosomes by flow cytometry. Proc Natl Acad Sci USA 1981;78:7727–7731.
21. van den Engh GJ, Trask BJ, Gray JW. The binding kinetics and interaction of DNA fluorochromes used in the analysis of nuclei and chromosomes by flow cytometry. Histochemistry 1986;84(4–6):501–508.
22. van den Engh G, Trask B, Lansdorp P, Gray J. Improved resolution of
flow cytometric measurements of Hoechst- and chromomycin-A3stained human chromosomes after addition of citrate and sulfite.
Cytometry 1988;9:266–270.
23. van den Engh GJ, Trask BJ, Gray JW, Langlois RG, Yu LC. Preparation
and bivariate analysis of suspensions of human chromosomes. Cytometry 1985;6:92–100.
24. Yu LC, Aten J, Gray J, Carrano AV. Human chromosome isolation from
short-term lymphocyte culture for flow cytometry. Nature 1981;
293(5828):154,155.
25. Rabbitts P, Impey H, Heppell-Parton A, Langford C, Tease C, Lowe N,
Bailey D, Ferguson-Smith M, Carter N. Chromosome specific paints
from a high resolution flow karyotype of the mouse. Nat Genet 1995;
9:369–375.
26. Cram LS, Bartholdi MF, Ray FA, Travis GL, Kraemer PM. Spontaneous
neoplastic evolution of Chinese hamster cells in culture: Multistep
progression of karyotype. Cancer Res 1983;43:4828–4837.
27. Blumenthal AB, Dieden JD, Kapp LN, Sedat JW. Rapid isolation of
metaphase chromosomes containing high molecular weight DNA.
J Cell Biol 1979;81:255–259.
28. Wray W, Stubblefield E. A new method for the rapid isolation of chromosomes, mitotic apparatus, or nuclei from mammalian fibroblasts at
near neutral pH. Exp Cell Res 1970;59:469–478.
29. Buys CH, Koerts T, Aten JA. Well-identifiable human chromosomes
isolated from mitotic fibroblasts by a new method. Hum Genet
1982;61:157–159.
30. Wray W. Isolation of metaphase chromosomes with high molecular
weight DNA at pH 10.5. Methods Cell Biol 1973;6:307–315.
31. Pelta J, Livolant F, Sikorav JL. DNA aggregation induced by polyamines
and cobalthexamine. J Biol Chem 1996;271:5656–5662.
32. Tabor H. The protective effect of spermine and other polyamines
against heat denaturation of deoxyribonucleic acid. Biochemistry
1962;1:496–501.
33. Wilson RW, Bloomfield VA. Counterion-induced condesation of deoxyribonucleic acid. A light-scattering study. Biochemistry 1979;18:
2192–2196.
34. Widom J, Baldwin RL. Cation-induced toroidal condensation of DNA
studies with Co31(NH3)6. J Mol Biol 1980;144:431–453.
35. Manning GS. The molecular theory of polyelectrolyte solutions with
applications to the electrostatic properties of polynucleotides. Q Rev
Biophys 1978;11:179–246.
36. Bloomfield VA. DNA condensation by multivalent cations. Biopolymers 1997;44:269–282.
37. Raspaud E, Olvera de la Cruz M, Sikorav JL, Livolant F. Precipitation of
DNA by polyamines: A polyelectrolyte behavior. Biophys J 1998;74:
381–393.
38. Gray JW, Cram LS.Flow karyotyping and chromosome sorting. In:Melamed MR,Lindmo T,Mendelsohn ML, editors.Flow Cytometry and Sorting,2nd ed.New York:Wiley-Liss; 1990. pp 503–529.
39. Gray JW, Dean PN, Fuscoe JC, Peters DC, Trask BJ, van den Engh GJ,
Van Dilla MA. High-speed chromosome sorting. Science 1987;
238(4825):323–329.
40. Behr W, Honikel K, Hartmann G. Interaction of the RNA polymerase
inhibitor chromomycin with DNA. Eur J Biochem 1969;9:82–92.
41. Langlois RG, Jensen RH. Interactions between pairs of DNA-specific
fluorescent stains bound to mammalian cells. J Histochem Cytochem
1979;27:72–79.
42. Latt SA, Sahar E, Eisenhard ME, Juergens LA. Interactions between
pairs of DNA-binding dyes: Results and implications of chromosome
analysis. Cytometry 1980;1:2–12.
Cytometry Part A DOI 10.1002/cyto.a