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Technical Advance |
From the Departments of Pathology*
and
Surgery,
Yamaguchi University School of
Medicine, Ube; and the National Institute of Bioscience and Human
Technology,
Tsukuba, Ibaraki, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1,000 cells were placed on each
spot. To demonstrate the usefulness of the cell array, we
measured nuclear DNA content using laser-scanning cytometry for DNA
ploidy analysis in nine human tumor cell lines and normal lymphocytes.
Combining the cell array with laser-scanning cytometry allows not only
measurement of nuclear DNA content for 50 samples but also easy
comparison of DNA ploidy among the samples in a single experiment. In
addition, we used the cell array for fluorescence in
situ hybridization using a DNA probe specific for the
pericentromeric region of chromosome 11.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this brief communication, we present a novel but simple device that facilitates not only immunohistochemistry and FISH but also measurement of DNA ploidy across multiple samples in a single experiment. This cell array device makes it possible to analyze 50 tumor specimens on a single slide. We describe the potential impact of the cell array device for measuring nuclear DNA content by laser scanning cytometry (LSC). To further demonstrate the uses of the cell array device, we perform FISH using DNA centromere.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A glass slide was specially designed for analysis of multiple
samples as shown in Figure 1
. A 30
x 16-mm square area on a glass slide was covered with black printing
ink without significant autofluorescence. In the area, 50 (10 x
5) 2-mm diameter spots were arranged at 1-mm intervals. The surface of
each spot was coated with triethoxyaminopropylsilane to prevent
desquamation of cells from the slide glass. (Detailed information
concerning the glass slide with spots was obtained from Matsunami Glass
Industries Ltd., Kishiwada, Japan). The numbers (1 to 10) and
letters (A to E) were printed along the margins of the black area to
identify the position of each spot.
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We used human peripheral blood lymphocytes and nine human tumor cell lines; U87MG, U105G, DBTRG65MG, U373MG, GI-1, SW1083, NB3911, HS683, and U251. The tumor cell lines were grown in Dulbecco’s modified Eagle medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% calf serum at 37°C in a humidified atmosphere of 5% CO2 in air. The cells were harvested with 0.25% trypsin in phosphate-buffered saline (PBS), then washed and resuspended in PBS, and fixed in 70% ethanol followed by 100% ethanol. Lymphocytes from peripheral blood were isolated by Ficoll-Hypaque, washed in PBS and fixed in 70% ethanol followed by 100% ethanol.
Cell Array Preparation
Drop cell suspensions were put as five spots on the glass slide
with a micropipette and then air-dried to investigate the difference in
DNA ploidy among spots of cells of the same type. The density of cells
in a spot was adjusted manually using a microscope. Each spot contained
800 to 1,000 cells because more than 1,000 cells per spot decreased the
number of countable cells. Peripheral lymphocytes were spotted in five
positions as controls for the cultured cells. A total of 10 cell types
was examined in the same glass slide. It took 1 hour to prepare one
array slide.
DNA Ploidy Analysis by LSC
The slides were treated with RNase (1 mg/ml; Sigma) for 30 minutes at room temperature and were then dipped in a solution of propidium iodide (25 µg/ml; Sigma). Coverslips were placed on the slides and sealed with nail polish. DNA content was measured with a laser-scanning cytometer (LSC 101; Olympus Co., Tokyo, Japan) as described previously.11-14 At first, nuclear DNA content was measured for the area covered with black printing ink containing all spots. Namely, DNA content and cellular position data were stored for all cells in list mode. Overlapping nuclei were automatically excluded from the counting by special statistical filters.15 Subsequently, a DNA histogram was generated for each spot or group of spots and DNA ploidy was determined. Our device allows any combination of spots to be grouped and evaluated. The DNA index (DI) was calculated according to principles recommended by consensus.16 A DI equal to 1.0 indicates DNA diploidy.
FISH
To verify the reliability of our cell array system, we performed FISH using a Spectrum Green-labeled alphoid satellite DNA probe specific for the pericentromeric region of human chromosome 11 (D11Z1; Vysis, Inc., Downers Grove, IL), as previously described.17 Briefly, 10 µl of a hybridization mixture containing 1 µg/ml of salmon sperm DNA (Sigma Chemical Co.), 55% formamide, 2x standard saline citrate (SSC) (1x SSC is 0.15 mol/L NaCl and 15 mol/L Na citrate) and 10% dextran sulfate was heated in a water bath at 70°C for 5 minutes. The DNA mixture was applied to the slides, which were denatured at 70°C for 2 minutes, and they were then incubated overnight at 37°C in a moist chamber. The slides were transferred to a washing solution containing 50% formamide and 2x SSC at 45°C, and then the nuclei were counterstained by adding glycerol with propidium iodide (2 µg/ml; Sigma) and p-phenylenediamine dihydrochloride (1 µg/µl; Sigma Chemical Co.).
Scoring of Hybridization Signals
The number of hybridization signals in each nucleus was determined by observing more than 200 nuclei in each spot with an epifluorescence microscope equipped with a 100x oil immersion objective (Olympus Co.). The percentage of cells with different signal counts was determined for each spot. The modal number of individual chromosomes represented the number of chromosomes in the tumor cells for each spot and grouped spots.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We were typically able to count 500 cells (range, 350 to 800
per spot) when aggregated cells were excluded from counting. Increasing
the cell density in a spot resulted in overlapping cells, which reduced
the number of cells that could be counted. Repeated experiments
revealed that the optimal cell density was 800 to 1,000 cells per
spot.
Measurement of Nuclear DNA Content
Nuclear DNA content was measured by LSC for propidium
iodide-stained cells. A total of 35,000 to 40,000 cells was counted for
50 spots in LSC DNA measurements. A DNA histogram for each spot was
constructed by setting a gate window in the cytogram, an X-Y
scattergram, that represents the distribution of both spots on the
glass slide and cells in each spot (Figure 2)
. Approximately 1 hour was necessary
for scanning the 30 x 16-mm area where 50 spots were arranged.
The number of countable cells was 500 per spot. The DI of the DNA
histogram was determined easily without the addition of an internal
standard because spots containing normal lymphocytes were included on
the same slide. No difference in the position of the
G1/G0 peak was found among
DNA histograms for five spots containing normal lymphocytes (Figure 2)
.
DI was the same for spots containing the same cell line. The positions
of the G1/G0 peak in DNA
histograms from lymphocytes and tumor cells differed distinctly (Figure 3)
. The DI of each cell line is
summarized in Table 1
. DI was easily
compared among cell lines by delineating a gate window so as to
encompass the spots of interest. It was also possible to combine two or
more spots into one using the gating procedure, and accordingly spots
consisting of the same type of cells grouped to form a single DNA
histogram. The increase in the number of spots containing the same type
of cells made each DNA histogram distinct (Figure 2)
. In this study,
the coefficient of variation was 5%.
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FISH using a DNA probe specific for the chromosome 11 centromere
revealed the chromosome copy number in tumor cells and control
lymphocytes. On average, 88% (range, 86 to 90%) of lymphocytes had
two signals in every spot, and lymphocytes with >3 signals were
virtually not detected. In tumor cell lines, the number of signals from
chromosome 11 was cell-dependent. The modal chromosome number was 4
with great intercellular variation in most of cell lines (Table 1)
.
There was no significant difference in modal chromosome number among
spots of the same cell lines including the lymphocyte controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cell array device presented here addresses the requirements mentioned above as well as functioning as a tissue microarray. However, our cell array system has advantages and disadvantages when compared to the tissue microarray. The cell array data provides no information about histological structures, which are available from the tissue microarray, but the cell array allows rapid analysis of DNA ploidy in multiple specimens. The cell array also permits reliable interphase cytogenetic analysis using chromosome-specific DNA probes. In contrast, a tissue microarray may lead to erroneous interpretation of chromosomal aberrations because of truncated nuclei in the tissue sections. This could be a serious problem because it is difficult to judge whether intercellular variations in chromosome number are accurate or artifactual. Cell array can compensate the disadvantages of tissue microarray, and vice versa.
Usually, more cells yield more reliable cytometric analysis. There is
an inverse relationship between the size of the spots and the number of
spots on a glass slide. There are fewer samples on a cell array than on
a tissue microarray. Hundreds of cells are required for analysis of DNA
ploidy and the cell cycle. This is why the cell array device is
designed to have only 50 spots, and this is a difference between our
cell array and other systems. It may be possible to increase the number
of spots per area. For example, an array with 200 spots (spot size,
1-mm diameter; interspot distance, 1 mm) can be prepared. However, this
is impractical because the number of cells within a smaller spot may be
insufficient for DNA ploidy analysis (data not shown). A 2-mm spot
diameter is a compromise between the density of spots and the number of
cells for DNA ploidy analysis. In this study, the maximum number of
countable cells was 1,000 per spot. However, an increase in cell
density in a spot is inevitably accompanied by an increase in the
number of overlapping cells, which reduces the number of countable
cells. Empirically, the optimum number of applied cells per spot is
1,000. However, the size of spots depends on analysis. In
experiments that require only a small number of cells for analysis, the
spot size can be reduced and the number of spots on a glass slide can
be increased as mentioned above. As a result, more samples are examined
in a single experiment.
The cell array device becomes more powerful when combined with LSC, which is a microscope-based cytometer with diverse analytical capabilities comparable to flow cytometry.15 In this study, LSC measurement of nuclear DNA content was done for nine cell lines and lymphocytes arrayed on a glass slide. This experiment proved that the combination of the cell array device with LSC is sophisticated and convenient for DNA ploidy analysis in many samples. Furthermore, use of the cell array device allows easy comparison of DNA ploidy among samples without inclusion of an internal standard because all samples stained equally with propidium iodide are on the same glass slide. In fact, no difference in the positions of the G1/G0 peaks was seen between DNA histograms generated from spots containing the same type of cells. This combination method permits reliable comparative studies of cellular characteristics among multiple samples and it is applicable to all samples measurable by LSC, such as for surface markers22 and intracellular substances.12 Cell array combined with LSC facilitates analysis of biomarkers in malignant tumors.
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Footnotes |
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Supported in part by the New Energy and Industrial Technology Development Organization (NEDO) of Japan.
Accepted for publication May 25, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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