81.
Versatility of analytical capabilities of laser scanning cytometry (LSC)
Grabarek, Jerzy; Darzynkiewicz, Zbigniew
Clinical and Applied Immunology Reviews,
04/2002, Letnik:
2, Številka:
2
Book Review, Journal Article
The microscope-based cytofluorometer laser scanning cytometer (LSC) combines many attributes of flow- and image-cytometry. Laser-excited fluorescence emitted from individual cells is measured at ...
multiple wavelengths, rapidly, with high sensitivity and accuracy. In this review the following analytical capabilities of LSC are discussed: (a) analysis of hyperchromicity of nuclear DNA to identify cell types that differ in chromatin condensation, mitotic or apoptotic cells; (b) topographic distribution of fluorescence within the cell, e.g., cytoplasm vs
. nucleus, nucleoplasm vs
. nucleolus, translocation of regulatory molecules such as NF-κB, p53, Bax; (c) analysis of micronuclei in mutagenicity assays; (d) fluorescence in situ hybridization (FISH); (e) morphometry and enumeration of nucleoli; (f) analysis of progeny of individual cells in clonogenicity assay; (g) cell immunophenotyping; (h) relocation of the measured cell for visual examination, imaging or sequential analysis using different immunochemical or cytochemical stains, or genetic probes; (i) analysis of in situ enzyme kinetics and other time resolved processes; (j) analysis of tissue section architecture. Other advantages and limitations of LSC are discussed and compared with flow cytometry.
več
Dostopno za:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
82.
Dostopno za:
NUK, UL, UM, UPUK
83.
Exposure of cells to static magnetic field accelerates loss of integrity of plasma membrane during apoptosis
Teodori, Laura; Grabarek, Jerzy; Smolewski, Piotr ...
Cytometry,
1 November 2002, Letnik:
49, Številka:
3
Journal Article
Background
Much attention is being paid to the biologic effects of magnetic fields (MFs). Although MFs enhance tumorigenesis, they are neither mutagenic nor tumorigenic. The mechanism of their ...
tumorigenic effect has not been elucidated.
Methods
To investigate the effect of MFs on apoptosis in HL‐60 cells, we exposed the cells to static MFs of 6 mT generated by a magnetic disk of known intensity. Apoptosis was triggered by the DNA topoisomerase I inhibitor, camptothecin (CPT). Activation of caspases in situ using the fluorochrome‐labeled inhibitor (FLICA) method and determination of plasma membrane integrity by excluding propidium iodide (PI) were measured by both laser scanning cytometry (LSC) and flow cytometry (FC). LSC and FC identified cells at three sequential stages of their demise: early apoptosis (cells with activated caspases and PI negative); late apoptosis (cells with activated caspases but unable to exclude PI); secondary necrosis (cells with apoptotic morphology no longer stained with FLICA, not excluding PI).
Results
MF alone did not induce any apoptogenic or necrogenic effect. CPT exposure led to the sequential appearance of apoptotic cells. In the presence of CPT and MF, the overall proportion of cells undergoing apoptosis was not significantly changed. However, we consistently observed a significant increase in the frequency of late apoptotic/necrotic cells when compared with samples treated with CPT alone (P < 0.001), as well as a decrease in the percentage of early apoptotic cells (P = 0.013). The data obtained by FC and LSC were consistent with each other, showing a similar phenomenon.
Conclusion
Whereas MF alone or with CPT did not affect overall cell viability, it accelerated the rate of cell transition from apoptosis to secondary necrosis after induction of apoptosis by the DNA‐damaging agent, CPT. Modulation of the kinetics of the transition from apoptosis to secondary necrosis by MF in vivo may play a role in inflammation and tumorigenesis. Cytometry 49:113–118, 2002. © 2002 Wiley‐Liss, Inc.
več
Dostopno za:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
84.
Detection of in situ activation of transglutaminase during apoptosis: Correlation with the cell cycle phase by multiparameter flow and laser scanning cytometry
Grabarek, Jerzy; Ardelt, Barbara; Kunicki, Jan ...
Cytometry (New York, N.Y.),
1 October 2002, 2002-Oct-01, 2002-10-00, Letnik:
49, Številka:
2
Journal Article
Background
One of the hallmarks of apoptosis is activation of tissue transglutaminase (Tgase; also called transglutaminase type 2 TGase 2). Its activation causes cross‐linking of cytoplasmic ...
proteins, making them insoluble and presumably less immunogenic. Several biochemical and cytochemical methods to detect activity of TGase 2 exist, but none has been adapted for multiparameter flow or image cytometry.
Methods
Apoptosis of HL‐60 or U‐937 leukemic cells was induced by camptothecin, tumor necrosis factor α, hyperthermia, or the cytotoxic RNase onconase. Two different approaches to detect TGase 2 activation were developed: (a) the unfixed cells were treated with 4′,6′‐diamidino‐2‐phenylindole, and sulforhodamine 101 in solutions of nonionic detergents; (b) the TGase 2 substrate fluoresceinated polyamine cadaverine (F‐CDV) was administered into the cultures for several hours before cell harvesting. The cells were then fixed and their DNA counterstained with propidium. Cellular fluorescence was measured by flow or laser scanning cytometry.
Results
(a) Exposure of nonapoptotic cells to detergents caused their full lysis, resulting in preparation of isolated nuclei devoid of cytoplasm. Conversely, the cross‐linking of cytoplasmic protein by activated TGase 2 in apoptotic cells provided resistance to detergents: the nuclei or nuclear (chromatin) fragments of apoptotic cells remained attached to the cytoplasmic protein, embedded within the proteinaceous “shell.” Such cells were identified by their high protein content: intensity of fluorescence after staining with the protein fluorochrome sulforhodamine 101 was markedly higher than that of isolated nuclei. (b) Activation of TGase 2 was also detected by virtue of intense cell labeling with fluoresceinated polyamine cadaverine. Interestingly, in many cells apoptosis progressed without evidence of activation of TGase 2, suggesting that this event may not be a prerequisite for completion of apoptosis.
Conclusions
Activation of TGase 2 can be detected simply by cell resistance to detergents or in situ reactivity with F‐CDV. Both methods allow one to correlate activation of TGase 2 with the cell cycle position. However, because activation of TGase 2 is not always detected during apoptosis, the lack of the activation cannot be considered a marker of nonapoptotic cells. Hence, an apoptotic index based solely on TGase 2 activation may underestimate incidence of apoptosis. Cytometry 49:83–89, 2002. © 2002 Wiley‐Liss, Inc.
več
Dostopno za:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
85.
Bivariate analysis of cellular DNA versus RNA content by laser scanning cytometry using the product of signal subtraction (differential fluorescence) as a separate parameter
Smolewski, Piotr; Grabarek, Jerzy; Kamentsky, Louis A. ...
Cytometry,
1 September 2001, 2001-Sep-01, 2001-09-01, 20010901, Letnik:
45, Številka:
1
Journal Article
Background
The cytometric methods of bivariate analysis of cellular RNA versus DNA content have limitations. The method based on the use of metachromatic fluorochrome acridine orange (AO) requires ...
rigorous conditions of the equilibrium staining whereas pyronin Y and Hoechst 33342 necessitate the use of an instrument that provides two‐laser excitation, including the ultraviolet (UV) light wavelength.
Methods
Phytohemagglutinin (PHA)‐stimulated human lymphocytes were deposited on microscope slides and fixed. DNA and double‐stranded (ds) RNA were stained with propidium iodide (PI) and protein was stained with BODIPY 630/650‐X or fluorescein isothiocyanate (FITC). Cellular fluorescence was measured with a laser scanning cytometer (LSC). The cells were treated with RNase A and their fluorescence was measured again. The file‐merge feature of the LSC was used to record the cell PI fluorescence measurements prior to and after the RNase treatment in list mode, as a single file. The integrated PI fluorescence intensity of each cell after RNase treatment was subtracted from the fluorescence intensity of the same cell measured prior to RNase treatment. This RNase‐specific differential value of fluorescence (differential fluorescence DF) was plotted against the cell fluorescence measured after RNase treatment or against the protein‐associated BODIPY 630/650‐X or FITC fluorescence.
Results
The scattergrams were characteristic of the RNA versus DNA bivariate distributions where DF represented cellular ds RNA content and fluorescence intensity of the RNase‐treated cells, their DNA content. The distributions were used to correlate cellular ds RNA content with the cell cycle position or with protein content.
Conclusions
One advantage of this novel approach based on the recording and plotting of DF is that only the RNase ‐specific fraction of cell fluorescence is measured with no contribution of nonspecific components (e.g., due to the emission spectrum overlap or stainability of other than RNA cell constituents). Another advantage is the method's simplicity, which ensues from the use of a single dye, the same illumination, and the same emission wavelength detection sensor for measurement of both DNA and ds RNA. The method can be extended for multiparameter analysis of cell populations stained with other fluorochromes of the same‐wavelength emission but targeted (e.g., immunocytochemically) for different cell constituents. Cytometry 45:73–78, 2001. © 2001 Wiley‐Liss, Inc.
več
Dostopno za:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
86.
Morphometry of nucleoli and expression of nucleolin analyzed by laser scanning cytometry in mitogenically stimulated lymphocytes
Gorczyca, Wojciech; Smolewski, Piotr; Grabarek, Jerzy ...
Cytometry (New York, N.Y.),
1 November 2001, 2001-Nov-01, 2001-11-01, Letnik:
45, Številka:
3
Journal Article
Background
Various attributes of nucleoli, including abundance of the nucleolar product (rRNA), correlate with cell‐proliferative status and are useful markers for tumor diagnosis and prognosis. ...
However, there is a paucity of methods that can quantitatively probe nucleolus. The aim of the present study was to utilize the morphometric capacity of the laser scanning cytometer (LSC) to analyze nucleoli and measure expression of the nucleolar protein nucleolin (NCL) in individual cells and correlate it with their state of proliferation.
Materials and Methods
Human lymphocytes were mitogenically stimulated, and at different time points their nucleoli were detected immunocytochemically using NCL Ab. The frequency of nucleoli per nucleus, their area, and the level of expression of NCL, separately in the nuclear and nucleolar compartments, were estimated in relation to the G0 to G1 transition and the cell cycle progression.
Results
During the first 24 h of stimulation, when the cells underwent G0 to G1 transition, their RNA content was increased nearly 8‐fold, the level of NCL per nucleus also increased 8‐fold, the NCL per nucleolus increased 12‐fold, nucleolear area increased 3‐fold, and NCL/nucleolar area increased nearly 4‐fold. During the subsequent 24–48 h of stimulation, when cells were progressing through S, G2, and M and reentering the next cycle, the number of nucleoli per nucleus was increased and a massive translocation of NCL from nucleoli to nucleoplasm was observed; its overall level per nucleus, however, still remained high, at 6‐fold above of that of G0 cells.
Conclusions
While high expression of NCL in the nucleolar compartment correlates with the rate of rRNA accumulation in the cell and is a sensitive marker of the G0 to G1 transition, the cells progressing through the remainder of the cycle are better distinguished from G0 cells by high overall level of NCL within the nucleus. Such an analysis, when applied to tumors, may be helpful in obtaining the quantitative parameters related to the kinetic status of the tumor‐cell population and tumor prognosis. The capability of LSC to measure the protein translocation between nucleolus and nucleoplasm can be used to study the function and regulatory mechanisms of other proteins that reside in these compartments. Cytometry 45:206–213, 2001. © 2001 Wiley‐Liss, Inc.
več
Dostopno za:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
87.
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