Microscope‐based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data

Kamentsky, Louis A.; Kamentsky, Lee

doi:10.1002/cyto.990120502

Cited by 226 publications

(156 citation statements)

References 9 publications

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“…The applicability of 7-amino actinomycin D (7-AMD), which offers certain advantages over PI as a DNA fluorochrome in combination with BODIPY-labeled deoxynucleotides ( 16), has also been tested. Furthermore, the utility of a recently developed microscope-based multiparameter laser scanning cytometer, the CompuCytometer (19), for the analysis of labeled cells was explored. We also demonstrate that preincubation of cells which incorporated BrdUrd with Hoechst 33258, by increasing their sensitivity to photolysis, raised the number of DNA strand breaks and thereby increased the sensitivity of their detection by the SBIP methodology.…”

mentioning

confidence: 99%

Single‐step procedure for labeling DNA strand breaks with fluorescein‐ or Bodipy‐conjugated deoxynucleotides: Detection of apoptosis and bromodeoxyuridine incorporation

et al. 1995

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The methods of in situ labeling of DNA strand breaks have been used to identify apoptotic cells and/or DNA replicating cells. While discrimination of apoptotic cells is based on the inherent presence of numerous DNA strand breaks in their chromatin, DNA proliferating cells can be discriminated by the selective DNA strand break induction by photolysis (SBIP) methodology at the sites that contain incorporated bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd). In both instances, DNA strand breaks are labeled with biotin-or digoxygenin-conjugated deoxynucleotides, preferably in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase; fluorescein tagged avidin (streptavidin) or digoxygenin antibody is used in the second step of the reaction. In the present study, DNA strand break labeling was simplified by using directly labeled deoxynucleotides, in a single-step reaction. Cell fluorescence was measured by flow cytometry as well as by a microscope-based laser scanning multiparameter cytometer. Apoptotic cells in HL-60 cultures treated with camptothecin or in primary cultures of non-Hodgkin's lymphoma cells treated with prednisolone were easily identifled utilizing BODIPY-conjugated dUTP (BdUTP). Apoptotic cells were also recognized using fluorescein-conjugated dUTP or dATP, although the discrimination was more pronounced with B-dUTP. The single-step procedure, requiring fewer centrifugation steps, resulted in less cell loss compared to the two-step cell labeling technique. The cells, labeled with BrdUrd for 30 min or 1 h, could also be identified in a single-step reaction; under the present conditions, better discrimination (signalto-noise ratio) was obtained measuring cell fluorescence with the microscope cytometer compared to cell analysis by flow cytometery. Preincubation of cells with Hoechst 33258 prior to ultraviolet (W) illumination, to enhance the photolysis, improved detection of cells which incorporated BrdUrd. The morphology of cells subjected to SBIP was excellent, allowing visualization of distinct DNA replication points. Because, unlike the immunocytochemical methods used to detect BrdUrd incorporation, the SBIP methodology does not require DNA denaturation by heat or acid, nuclear proteins are expected to remain undenatured in situ, allowing one to study colocalization of various constituents, detected immunocytochemically, at the DNA replication points. 0 1995 Wiley-Liss, h c . -14,20,28). Fluoresceinated avidin or digoxygenin antibodies are then used in the second step of the reaction to visualize the DNA strand breaks. Counterstaining of DNA with propidium iodide (PI) followed by bivariate analysis of the data allows one to relate apoptosis to cell cycle position (14) or DNA ploidy (3).A method of detection of bromodeoxyuridine ( BrdUrd) or iododeoxyuridine (IdUrd) incorporated into DNA was recently developed in our laboratory which also is based on the in situ labeling of DNA strand breaks (23). In this method [strand breaks induced by photolysis (SBIP)], DNA strand breaks a...

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confidence: 99%

Single‐step procedure for labeling DNA strand breaks with fluorescein‐ or Bodipy‐conjugated deoxynucleotides: Detection of apoptosis and bromodeoxyuridine incorporation

et al. 1995

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“…The instrumentation and the software of the laser scanning cytometer (CompuCyte, Cambridge, MA), as well as its clinical applications, were described elsewhere in detail (14,15). On this slide-based instrument, in principle any sample immobilized on a microscope slide can be analyzed: the laser scanning cytometer used in this study was equipped with an Argon (Ar)-laser (488 nm) and a Helium-Neon-laser (633 nm), and with four photomultipliers with standard optical filters and mirrors for detection of FITC, PE, PI, and APC/Cy5 (3).…”

Section: Laser Scanning Cytometry (Lsc)mentioning

confidence: 99%

Quantitative histology by multicolor slide‐based cytometry

et al. 2004

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BackgroundIn lymphatic organs, the quantitative analysis of the spatial distribution of leukocytes by tissue cytometry would give relevant information about alterations during diseases (leukemia, HIV, AIDS) and their therapeutic regimen, as well as in experimental settings.MethodsWe have developed a semiautomated analysis method for laser scanning cytometry (LSC) termed “multiple thresholding,” which is suitable for archived or fresh biopsy material of human lymph nodes and tonsils. Sections are stained with PI for nuclear DNA and up to four antigens using direct or indirect immunofluorescence staining. Measurement is triggered on DNA‐fluorescence (argon laser, Ar) or on specific cell labeling. Due to the heterogeneity of cell density, measurements are performed repeatedly at different threshold levels (low threshold: regions of low cellular density, germinal center; high threshold: dense regions, mantle zone). Data are acquired by single‐ (Ar) or dual‐laser excitation (Ar‐HeNe) in order to analyze single‐ (FITC) up to four‐color (FITC/PE/PECy5/APC) stained specimen.ResultsPercentage and cellular density of cell‐subsets is quantified in different microanatomical regions of the specimen. These data were highly correlated with manual scoring of identical specimens (r2 = 0.96, P < 0.0001). With LSC, semiautomated operator‐independent immunophenotyping in tissue sections of lymphatic organs with up to three antibodies simultaneously is possible.ConclusionsWe expect this tissue cytometric approach to yield new insight into processes during diseases and help to quantify the success of therapeutic interventions. © 2004 Wiley‐Liss, Inc.

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Dna Ploidy Analysis By Lscmentioning

confidence: 99%

The Development of a Cell Array and Its Combination with Laser-Scanning Cytometry Allows a High-Throughput Analysis of Nuclear DNA Content

Oode

Furuya

Harada

et al. 2000

The American Journal of Pathology

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We developed a cell array device for analyzing cellular characteristics such as DNA ploidy, numerical chromosomal aberrations, and antigen expression in multiple specimens in a single experiment. Fifty (10 ؋ 5) spots, 2 mm in diameter, were arrayed in an area of 30 ؋ 16 mm on a glass slide, and ϳ1,000 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. Examination of cellular properties has become increasingly important in the diagnosis and treatment of malignant tumors, and various in situ methods are currently available. Immunohistochemistry is a routine procedure for histopathological examination of tumors. Fluorescence in situ hybridization (FISH), which is applicable to interphase cells and metaphase spreads, is widely used in both research and clinical analyses. FISH provides direct information for the diagnosis of chromosomal aberrations. In both immunohistochemistry and FISH, each marker is examined individually for each sample at a time. When many samples have to be examined, such a one-by-one procedure is laborious, cumbersome, and time-consuming. Conventional methods require considerable amounts of antibody or DNA probe for each experiment. A novel method that allows efficient analysis of a large number of specimens would reduce both cost and time of analysis. To address this issue, a tissue microarray system that permits immunohistochemical and FISH analysis of hundreds of specimens in a single experiment was developed. [1][2][3][4] However, this method does not always permit precise analysis of gene (chromosome) copy number aberrations because nuclei are frequently truncated in tissue sections. Although DNA ploidy is one of the useful parameters for estimating the biological characteristics of tumors, 5-10 truncated nuclei in tissue microarrays hamper the precise measurement of nuclear DNA content. Alternative technology is necessary for a multiparameter analysis including DNA ploidy determination in a number of specimens. Usually, at least two experiments are made to determine DNA ploidy for each specimen. After the initial experiment, the addition of an internal standard, such as lymphocytes, is required to determine which peak represents diploidy in a DNA histogram. More convenient methods are necessary for clinical application of DNA ploidy.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...

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Microscope‐based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data

Cited by 226 publications

References 9 publications

Single‐step procedure for labeling DNA strand breaks with fluorescein‐ or Bodipy‐conjugated deoxynucleotides: Detection of apoptosis and bromodeoxyuridine incorporation

Single‐step procedure for labeling DNA strand breaks with fluorescein‐ or Bodipy‐conjugated deoxynucleotides: Detection of apoptosis and bromodeoxyuridine incorporation

Quantitative histology by multicolor slide‐based cytometry

The Development of a Cell Array and Its Combination with Laser-Scanning Cytometry Allows a High-Throughput Analysis of Nuclear DNA Content

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