Essential Cytometry Methods
1. The first to report on rapid induction of transcriptional activity during mitogenic stimulation of noncycling cells (G0 lymphocytes) preceding their proliferation.
(Darzynkiewicz Z, Krassowski T, Skopinska E: Effect of phytohemagglutinin on synthesis of "rapidly labeled" ribonucleic acid in human lymphocytes. Nature 207:1402,1965).
2. The first to use "dry autoradiography" to detect subcellular localization of the soluble enzyme (folate reductase) in tissues.
(Darzynkiewicz Z, Rogers AW, Barnard EA, Wang DH, Werkheiser WC. Autoradiography with tritiated methotrexate and the cellular distribution of folate reductase. Science 151:1528-1530, 1966).
3. The first to use conventional and "track" autoradiography to estimate location and number of acetylcholinesterase and serine protease(s) molecules in megakaryocytes, neuromuscular junctions and mast cells.
(Darzynkiewicz Z, Rogers AW, Barnard, E.A.: The number of acetyl-cholinesterase molecules in the rat megakaryocyte. J Histochem Cytochem 14:915-922, 1966; Rogers AW. Darzynkiewicz Z, Barnard EA, Salpeter MM. Number and location of acetylcholinesterase molecules at motor endplates of the mouse. Nature 210:1003 1006, 1966; Darzynkiewicz Z., Barnard, E.A.: Specific proteases of the rat mast cell. Nature 213:1198-1202, 1967).
4. The first to demonstrate that size ("dry mass") of quiescent (G0) cells (lymphocytes) is many-fold lower than of the mitogen-stimulated cells and thus can be used as a marker to identify G0 cells.
(Darzynkiewicz Z, Dokov V, Pienkowski M. Dry mass of lymphocytes during transformation after stimulation by phytohemagglutinin. Nature 214:1265-1266, 1967).
5. Adapted "dry autoradiography" to detect Na+ ions in individual cells.
(Darzynkiewicz Z, Komender J. Autoradiographic detection of sodium-24 ions in single cells. J Histochem Cytochem 15:605-607, 1967).
6. The first to report on chromatin changes during normal and abnormal spermatogenesis reflected by altered accessibility of DNA to actinomycin D.
(Darzynkiewicz Z, Gledhill BL, Ringertz NR. Changes in deoxyribo-nucleoprotein during spermiogenesis in the bull. 3H-Actinomycin D binding. Exp Cell Res 58:435-438, 1969.)
7. The first to demonstrate that HEPES buffer (previously used only to buffer cell-free solutions) instead of bicarbonate buffer can be used in tissue culture to maintain proper pH.
(Darzynkiewicz Z, Jacobson B. HEPES-buffered media in lymphocyte cultures. Proc Soc Exp Biol Med 136:387-393, 1971).
8. The first to report that hyaluronate suppresses lymphocyte stimulation and to propose that intercellular matrix modulates lymphoproliferative response to mitogens or antigens.
(Darzynkiewicz Z, Balazs EA. Effect of connective tissue intercellular matrix on lymphocyte stimulation. I. Suppression of lymphocyte stimulation by hyaluronic acid. Exp Cell Res 66:113-123, 1971).
9. The first to demonstrate that DNA repair (unscheduled DNA synthesis) can be induced in cells that lack the repair enzymes (hen erythrocytes, Xeroderma Pigmentosum cells) when they are fused with heterologous cells to form heterokaryons, in which the partner cell provide the DNA repair machinery. This finding, emphasized by Editorial in Nature (240:12,1972) provided experimental model to reveal Xeroderma Pigmentosum complementary groups and was first to demonstrate activity of UV endonuclease across the species (mouse or hen vs human).
(Darzynkiewicz Z, Chelmicka-Szorc E. Unscheduled DNA synthesis in hen erythrocyte nuclei reactivated in heterokaryons. Exp Cell Res 74:131-139, 1972; Darzynkiewicz Z, Chelmicka-Szorc E, Arnason BGW. U.V.-induced DNA synthesis in nuclei of Xeroderma Pigmento sum cells in heterokaryons. Exp Cell Res 74:602-608, 1972).
10. The first to reveal that cell ability to repair DNA through unscheduled DNA synthesis ceases at late stages of spermatogenesis and sperm maturation.
(Gledhill,BL, Darzynkiewicz Z. Unscheduled DNA synthesis during mammalian spermatogenesis in response to U.V. irradiation. J Exp Zool 183:375-382, 1973).
11. The first to demonstrate that activation of hen erythrocyte nuclei in heterokaryons is prevented by serine protease inhibitors, suggesting involvement of proteolytic activity in the reactivation process.
(Darzynkiewicz Z, Chelmicka-Szorc E, Arnason BGW. Chick erythrocyte nucleus reactivation in heterokaryons: Suppression by inhibitors of proteolytic enzymes. Proc Natl Acad Sci USA 71:644-647, 1974.)
12. Developed the assay for differential staining of DNA and RNA for cytometry exploiting metachromatic property of acridine orange.
(Darzynkiewicz Z, Traganos F, Sharpless T, Melamed MR. Conformation of RNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp Cell Res 95:143-153, 1975).
13. The first to demonstrate that quiescent G0 cells can be distinguished from cycling cells by their minimal RNA content detected by cytometry. This approach was accepted worldwide and the RNA-parameter is now considered one of the unquestionable markers of G0 cells.
(Darzynkiewicz Z, Traganos F, Sharpless T, Melamed MR.Lymphocyte stimulation: A rapid multiparameter analysis. Proc Natl Acad Sci USA 73:2881-2884, 1976) (cited over 440 times).
14. Developed the assay of susceptibility of DNA in situ to denaturation. This cytometric assay, which is worldwide accepted, allows one to distinguish cells based on the degree of chromatin condensation such as G0 from G1, G2 from M, or identify apoptotic cells.
(Darzynkiewicz., Traganos F, Sharpless T, Melamed MR. Cytofluorometric studies on conformation of nucleic acids in situ. II. Denaturation of DNA. J Histochem Cytochem 24:49-58, 1976). Darzynkiewicz, Z, Traganos F, Sharpless T, Melamed MR. Different sensitivity of DNA in situ in interphase and metaphase chromatin to heat denaturation. J Cell Biol 73:128-138, 1977.)
15. Developed cytometric approach to identify cycling cells based on incorporation of BrdU, which is detected via quenching of acridine orange fluorescence.
(Darzynkiewicz Z, Andreeff M, Traganos F, Sharpless T, Melamed MR. Discrimination of cycling and noncycling lymphocytes by BrdUrd-suppressed acridine orange fluorescence in a flow cytometric system. Exp Cell Res 115:31-35, 1978).
16. The first to demonstrate that the rate of cell progression through S or throughout whole cell cycle is correlated with cellular RNA content.
(Darzynkiewicz Z, Evenson D, Staiano-Coico L, Sharpless T, Melamed MR. Relationship between RNA content and progression of lymphocytes through the S phase of the cell cycle. Proc Natl Acad Sci USA 76:358-362, 1979; cited over 125 times; Darzynkiewicz Z, Evenson DP, Staiano-Coico L, Sharpless T, Melamed MR. Correlation between cell cycle duration and RNA content. J Cell Physiol 100:425-438, 1979).
17. Based on the observation that DNA in chromatin of abnormal sperm cells has higher propensity to DNA denaturation adapted the developed earlier cytometric assay of DNA denaturation (see point 14) to identify abnormal sperm cells. This assay ("sperm chromatin structure assay"; SCSA) was accepted by FDA and WHO and is now being used worldwide as the male fertility assay.
(Ringertz NR, Gledhill BL, Darzynkiewicz Z. Changes in deoxyri-bonucleoprotein during spermiogenesis in the bull. II. Sensitivity of DNA to heat denaturation. Exp Cell Res 62:204-218, 1970; Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210:1131-1133, 1980; cited over 270 times; U.S. Patent No. 4,559,309 Issued Dec. 17, 1985: "Flow Cytometry-Fluorescence Measurements for Characterizing Sperm").
18. Using his cytometric methods to concurrently measure RNA and DNA as well as to detect DNA denaturation identified 12 functionally distinct cell cycle subcompartments by multiparameter cytometry.
(Darzynkiewicz Z, Traganos F, Melamed MR. New cell cycle compartments identified by multiparameter flow cytometry. Cytometry 1:98-108, 1980; cited over 280 times; Darzynkiewicz Z, Sharpless T, Staiano-Coico L, Melamed MR. Subcompartments of the G1 phase of cell cycle detected by flow cytometry. Proc Natl Acad Sci USA 77:6696-6700, 1980; cited over 230 times).
19. The first to use rhodamine 123 as mitochondrial electrochemical potential probe in cytometry and to identify G0 cells as the cells with lower ability, compared to cycling cells, to bind this probe.
(Darzynkiewicz,, Staiano-Coico L, Melamed MR. Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation. Proc Natl Acad Sci USA 78:2383-2387, 1981, cited over 140 times; Darzynkiewicz Z, Traganos F, Staiano-Coico L, Kapuscinski J,.Melamed M.R. Interactions of rhodamine 123 with living cells studied by flow cytometry. Cancer Res 42:799-806, 1982; cited over 150 times).
20. The first to reveal that changes in heterogeneity of cell populations progressing through the cell cycle with respect to their size, RNA and protein content, also to demonstrate that due to asymmetric cytokinesis the heterogeneity increases after mitosis as well as to show that the cell populations become most uniform at the restriction point in G1 when the small cells are hold in progression until they "catch up" in growth of their size, with the larger ones.
(Darzynkiewicz Z, Crissman H, Traganos F, Stainkamp J. Cell heterogeneity during the cell cycle. J Cell Physiol 112:465-474, 1982; cited over 115 times).
20. The first to describe the cell cycle specific effects of tumor necrosis factor alpha.
(Darzynkiewicz, Z, Williamson B, Carswell EA, Old LJ. The cell cycle specific effects of tumor necrosis factor. Cancer Res 44:83-90, 1984; cited over 210 times).
21. The first to report that accessibility of DNA in situ to different fluorochromes varies depending on chromatin structure and therefore cell stainability with these dyes not always reflects DNA content (ploidy).
(Darzynkiewicz, Z., Traganos, F., Kapuscinski, J., Staiano-Coico, L., and Melamed, M.R.: Accessibility of DNA in situ to various fluorochromes: Relationship to chromatin changes during erythroid differentiation of Friend leukemia cells. Cytometry, 5:355-363, 1984; cited over 190 times).
22. The first to report that intercalating agents cause DNA condensation in solutions and in situ. This a novel observation that bears on mechanism of metachromatic DNA staining with various dyes, on mechanism of drug action, and has a potential to develop a method of separation of different oligonucleotides/nucleic acids based on differences in bases composition.
(Kapuscinski J, Darzynkiewicz Z. Condensation of nucleic acids by intercalating aromatic cations. Proc Natl Acad Sci USA 81:7368-7372, 1984; Darzynkiewicz Z, Traganos F, Kapuscinski J, Melamed MR. Denaturation and condensation of DNA in situ induced by acridine orange. Relationship to chromatin structure of Friend erythroleukemia cells. Cytometry, 6:195-207, 1985; U.S. Patent No. 4,801,550, issued Jan. 31, 1989 "Method for Separating and Analyzing Nucleic Acids").
23. The first to report that the new new antitumor drug mitoxantrone (novantrone) interacts with DNA by intercalation
(Kapuscinski J, Darzynkiewicz Z. Interactions of antitumor agents ametantrone and mitoxantrone (novantrone) with double-stranded DNA. Biochem Pharmacol 34:4203-4213, 1985.
Kapuscinski J, Darzynkiewicz Z, Traganos F, Melamed MR. Interactions of a new antitumor agent 1,4-dihydroxy-5-8-bis 2-(hydroxyethyl)amino)ethyl)amino)--9,10-anthracenedione with nucleic acids. Biochem Pharmacol 30:231-240, 1981; cited over 150 times).
24. The first to describe molecular bases of the mechanism of staining of DNA and RNA with pyronin Y.
(Kapuscinski J, Darzynkiewicz, Z. Interactions of pyronin Y (G) with nucleic acids. Cytometry 8:129-137, 1987;Darzynkiewicz Z, Kapuscinski J, Traganos F, Crissman HA. Application of pyronin Y (G) in cytochemistry of nucleic acids. Cytometry 8:138-145, 1987;.Traganos F, Crissman HA, Darzynkiewicz Z. Staining with pyronin Y detects changes in conformation of RNA during mitosis and hyperthermia of CHO cells. Exp Cell Res 179:535-544, 1988).
25. The first to report that the cytotoxic ribonuclease (Pannon) isolated from Xenopus has antitumor properties. This ribonuclease (currently named Onconase or Ranpirnase) is now in phase III clinical trials where it shows significant activity against malignant mesothelioma.
(Darzynkiewicz Z, Carter SP, Mikulski SM, Shogen K. Cytostatic and cytotoxic effects of Pannon, a novel anticancer agent. Cell Tissue Kinet 21:169-182, 1988; cited over 70 times).
26. The first to demonstrate that DNA topoisomerase I inhibitor camptothecin selectively induces apoptosis of DNA replicating cells.
(Del Bino G, Lassota P, Darzynkiewicz ,Z. The S-phase cytotoxicity of camptothecin. Exp Cell Res 294:27-35, 1991; cited over 155 times)
27. The first to show that caffeine is the interceptor of aromatic compounds and by heteroassociation modulates effects of several antitumor drugs.
(Traganos F, Kapuscinski J, Darzynkiewicz Z. Caffeine modulates the effects of DNA intercalating drugs in vitro: A flow cytometric and spectrophotometric analysis of caffeine interaction with Novantrone, doxorubicin, ellipticine and the doxorubicin analog, AD198, Cancer Res 51:3682-3689, 1991; Traganos, F., Kapuscinski, J., Gong, J., Ardelt, B., Darzynkiewicz, R.J., and Darzynkiewicz, Z.: Caffeine prevents apoptosis and cell cycle effects induced by camptothecin or topotecan in HL-60 cells. Cancer Res., 53: 4613-4618, 1993.)
28. The first to reveal that lovastatin arrests cells in G1 possibly by preventing the anchoring of c-ras in plasma membrane. This is the first demonstration that statins may have cytostatic properties or can be used to arrest and synchronize cells in the cell cycle.
(Jakóbisiak M, Bruno S, Skierski J, Darzynkiewicz Z. The cell cycle specific effects of lovastatin. Proc Natl Acad Sci USA 88:3628-3632, 1991; cited over 190 times).
29. The first to report that the protein kinases inhibitor staurosporine has different cytostatic effect on tumor (leukemia)- than on normal (lymphocyte)- cells and to suggest that staurosporine or similar kinase inhibitors should be used in conjunction with chemo- or radiotherapy to protect normal cells.
(Bruno S, Ardelt B, Skierski JS, Traganos F, Darzynkiewicz Z. Different effects of staurosporine, an inhibitor of protein kinases, on the cell cycle and chromatin structure of normal and leukemic lymphocytes. Cancer Res 52:470-473, 1992). Darzynkiewicz Z. Apoptosis in antitumor strategies: Modulation of cell cycle or differentiation. J Cell Biochem 58:151-159,1995; cited over 100 times).
30. Developed the assays to label DNA strand breaks with fluoresceinated deoxynucleotides in the reaction catalyzed by DNA polymerase or terminal deoxynucleotidyl transferase ("ISEL"; "TUNEL"). These assays are used worldwide to identify apoptotic cells, primarily by cytometry. Also was first to report that serine protease inhibitors prevent intenucleosomal DNA fragmentation during apoptosis
(Darzynkiewicz Z, Bruno S, Del Bino G, Gorczyca W, Hotz MA, Lassota P, Traganos F. Features of apoptotic cells measured by flow cytometry. Cytometry, 13:795-808, 1992; cited over 1,240 times; Gorczyca W, Bruno S, Darzynkiewicz RJ, Gong J, Darzynkiewicz, Z. DNA strand breaks occurring during apoptosis: Their early in situ detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. Int J Onc 1:639-648, 1992; cited over 210 times; Gorczyca W, Gong J, Darzynkiewicz Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 53:1945-1951, 1993; cited over 730 times).
31. The first to demonstrate that during chemotherapy leukemic cells are killed by the mechanism of apoptosis.
(Gorczyca W, Bigman K, Mittelman A, Ahmed T, Gong J, Melamed MR, Darzynkiewic, Z. Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia 7: 659-670, 1993; cited over 240 times; Li X, Gong J, Feldman E, Seiter K, Traganos F, Darzynkiewicz Z. Apoptotic cell death during treatment of leukemias. Leukemia and Lymphoma, 13: Suppl. 1, 65-70, 1994.).
32. The first to report the presence of a multitude of double-strand DNA breaks in human abnormal sperm cells and to propose that the apoptosis-like mechanism leads to elimination of defective sperm cells from reproductive pool. This assay is now used in different institutions to test male fertility.
(Gorczyca, W., Traganos, F., Jesionowska, H., Darzynkiewicz, Z.: Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells. Analogy to apoptosis of somatic cells. Exp Cell Res 207:202-205, 1993; cited over 130 times).
33. Developed the method to selectively extract fragmented DNA from apoptotic cells. This method is now widely used to analyze DNA fragmentation during apoptosis and to identify apoptotic cells as the cells with fractional DNA content ("sub-G1"; "hypodiploid") by cytometry.
(Gong J, Traganos F, Darzynkiewicz Z. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Analyt Biochem 218:314-319,1994, cited over 360 times).
34. Developed the method to reveal DNA replication based on the induction of DNA strand breaks by photolysis at the sites of BrdU incorporation (Strand Break-Induction by Photolysis; SBIP). Unlike the conventional method to detect the incorporated BrdU that requires acid or heat treatment the SBIP approach is fully compatible with the concurrent detection of surface or intracellular antigens. The kits based on this procedure (ABSOLUTE-STM) are provided by many vendors and widely used.
(Li X, Traganos F, Melamed MR. Darzynkiewicz Z. Detection of 5-bromo-2-deoxyuridine incorporated into DNA by labeling strand breaks induced by photolysis (SBIP). Int J Oncol 4:1157-1161, 1994; Li X, Melamed MR, Darzynkiewicz Z. Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color. Exp Cell Res 222: 28-37, 1996; U.S. Patent No. 5,747,258, Issued May 5, 1998 "Detection of Halogenated Precursors Incorporation by Selective Photolysis of Halogenate-Substituted Bases").
35. Developed the most sensitive method to detect DNA fragmentation that occurs during apoptosis, that is based on terminal transferase-mediated BrdU labeling of DNA strand breaks. The kits based on this variant of TUNEL assay (APO-BRDUTM), offered by many vendors, currently are the most widely used in analysis of DNA fragmentation during apoptosis.
(Li X, Darzynkiewicz, Z. Labeling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Proliferation 28: 571-579, 1995; cited over 120 times; U.S. Patent No. 5,912,126, Issued June 15, 1999, Methods for Labeling DNA Ends with Halogenated Nucleotides and Detecting the Same with Antibodies).
36. Developed the simplest, single-step method, to label DNA strand breaks that occur during apoptosis. The kits based on this procedure (APO-DIRECTTM), provided by many vendors, have also found wide utility.
(Li X, Traganos F, Melamed MR, Darzynkiewicz Z. Single step procedure for DNA strand break labeling. Detection of apoptosis and DNA replication. Cytometry 20:172-180, 1995).
37. The first to report that cells synchronized in the cell cycle by agents that prevent DNA replication show significant growth unbalance and have unscheduled expression of cyclins A, B and E. Such cell synchronization, thus, induces gross bias in studies of kinetics and molecular mechanisms controlling cell cycle progression.
(Gong J, Traganos F, Darzynkiewicz Z. Growth imbalance and altered expression of cyclins B1, A. E and D3 in MOLT-4 cells synchronized in the cell cycle by inhibitors of DNA replication. Cell Growth & Differentiation, 6: 1485-1493,1995).
38. The first to report that most tumor cell lines express cyclins D, A and B1 in an unscheduled manner. Specifically, D cyclins are expressed constitutively throughout all cell cycle, cyclin B1 is often expressed in G1 and cyclin E throughout all G1 phase. Such unscheduled cyclin expression may contribute to loss of control on cell cycle progression in cancer
(Darzynkiewicz Z, Gong J, Juan G, Ardelt B, Traganos, F.: Cytometry of cyclin proteins. Cytometry 25: 1-13, 1996; cited over 100 times).
39. Developed the method to separate nucleic acids labeled with BrdU (DNA) or BrU (RNA) by immuno-precipitation which makes it possible to isolate mRNA for analysis of the genes transcribed during the BrU pulse period.
(Haider SR, Juan G, Traganos F, Darzynkiewicz Z. Immuno-separation and immuno-detection of nucleic acids with halogenated nucleotides. Exp Cell Res 234: 498-506, 1997; US patent No. 6,417,343, issued July 9, 2002. "Physical Separation of Nucleic Acids by Antibodies to Halogenated Nucleotides").
40. The first to immunocytochemically monitor state of phosphorylation of proteins in individual cells by cytometry (Ser-10 histone H3 and hypophosphorylated pRB).
(Juan G, Traganos F, James WM, Ray JM, Roberge M, Sauve DM, Anderson H, Darzynkiewicz Z. Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis. Cytometry 32: 71-77, 1998; Juan G, Gruenwald S, Darzynkiewicz, Z. Phosphorylation of retinoblastoma susceptibility gene protein assayed in individual lymphocytes during their mitogenic stimulation. Exp Cell Res 239: 104-110, 1998; US patent No. 6,821740 issued November 23, 2004 " Flow cytometric methods for the concurrent detection of discrete functional conformations of pRB in single cells").
41. The first to measure kinetics of enzymatic reactions and fluorochrome uptake in the same individual cells by cytometry.
(Bedner E, Melamed MR, Darzynkiewicz Z. Time resolved kinetic reactions measured in individual cells by laser scanning cytometry (LSC) Cytometry 33: 1-9, 1998).
42. The first to measure, in the same cells, the attributes that can be measured only in live cells and correlate them with the attributes that require prior cell fixation and permeabilization to be measured. This approach was used to reveal that caspases activation during apoptosis may occur in the cells that have the mitochondrial potential still preserved.
(Li X, Darzynkiewicz Z. The Schrödinger's cat quandary in cell biology: Integration of live cell functional assays with measurements of fixed cells in analysis of apoptosis. Exp Cell Res 249: 404-412, 1999; Li X, Du L, Darzynkiewicz, Z. Caspases are activated during apoptosis independently of dissipation of mitochondrial electrochemical potential. Exp Cell Res 257: 290-297, 2000).
43. Developed simple assay to detect translocation of Bax to mitochondria during initiation step of apoptosis, based on measurement of maximal pixel intensity of Bax immunofluorescence.
(Bedner E, Li X, Kunicki J, Darzynkiewicz Z. Translocation of Bax to mitochondria during apoptosis measured by laser scanning cytometry. Cytometry 41: 83-88, 2000).
43. The first to report that the fluorochrome-labeled inhibitors of caspases (FLICA) can be used to detect apoptosis and most likely, to detect caspases activation. Collaborated with ONCOR Inc. and Immunochemistry Technologies, Inc. towards development of the FLICA probes.
(Bedner E, Smolewski P, Amstad P, Darzynkiewicz Z. Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp Cell Res 259: 308-313, 2000).
44. The first to reveal that during apoptosis DNA and RNA are segregated from each other and packaged into separate apoptotic bodies. Proposed that such segregation may facilitate degradation of nucleic acids after phagocytosis of apoptotic bodies.
(Halicka HD, Bedner E, Darzynkiewicz Z. Segregation of RNA and separate packaging of DNA and RNA in apoptotic bodies during apoptosis. Exp Cell Res 260: 248-255, 2000.
45. Developed an approach to stain cells by dye diffusion from gels that may be applicable as "liquidless" staining method at microgravity conditions in space.
(Smolewski P, Bedner E, Gorczyca W, Darzynkiewicz Z. "Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space. Cytometry 44: 355-360. 2001).
46. Developed an approach to semi-automatically measure micronucleation e.g. induced by genotoxic agents, by laser scanning cytometry.
(Smolewski P, Ruan Q, Vellon L, Darzynkiewicz Z. The micronuclei assay by laser scanning cytometry. Cytometry, 45: 19-26, 2001).
47. Collaborating with Immunochemistry Technologies, Inc., developed fluorochrome-tagged inhibitors/ligands for active serine proteases. The probes were designed to bind to serine proteases enzymatically active center and thus detect activation of these enzymes during apoptosis.
(Grabarek J, Du L, Johnson GL, Lee B, Phelps DJ. Darzynkiewicz Z: Sequential activation caspases and serine proteases (serpases) during apoptosis. Cell Cycle 1: 124-131, 2002;
Grabarek J, Darzynkiewicz Z. In situ activation of caspases and serine proteases during apoptosis detected by affinity labeling their enzyme active centers with fluorochrome-tagged inhibitors. Exp Hematol 30: 982-992, 2002).
48. Developed the cytometric method to detect activation of transglutaminase during apoptosis.
(Grabarek J, Ardelt B, Kunicki J, Darzynkiewicz Z. Detection of in situ activation of transglutaminase during apoptosis: correlation with the cell cycle phase by multiparameter flow- and laser scanning- cytometry. Cytometry 49: 83-89, 2002).
49. Proposed novel explanation for mechanisms of cytostatic/cytotoxic activity of ribonucleases such as Onconase, based on targeting RNAi.
(Ardelt B, Ardelt W, Darzynkiewicz Z. Cytotoxic ribonucleases and RNA interference (RNAi). Cell Cycle 2: 22-24, 2003.)
50. Developed in vitro model of wound healing adapted to laser scanning cytometry that can be used to study effects of exogenous agents on the healing (cell proliferation, migration). Using this method reported that hyaluronate facilitates healing of epithelial cells by decreasing incidence of apoptosis and enhancing cell migration).
(Haider AS, Grabarek J, Eng B, Pedraza P, Ferreri NR, Balazs EA, Darzynkiewicz Z. In vitro wound healing analyzed by laser scanning cytometry. Accelerated healing of epithelial cell monolayers in the presence of hyaluronate. Cytometry 53A:1-8, 2003; the article received "The best paper in CYTOMETRY 2003"award).
51. Developed the assay and was the first to estimate DNA damage (double-strand breaks) induced by DNA topoisomerase I and II inhibitors or UV light in individual cells by multiparameter cytometry (by measuring expression of Ser139-phosphorylated histone H2AX and phosphorylation of ATM on Ser1981) and correlate it with cell cycle phase and initiation of apoptosis (caspase-3 activation).
(Huang X, Traganos F, Darzynkiewicz Z. DNA damage induced by DNA topoisomerase I- and topoisomerase II- inhibitors detected by histone H2AX phosphorylation in relation to the cell cycle phase and apoptosis. Cell Cycle 2: 614-619; 2003; Halicka HD, Huang X, Traganos F, King MA, Dai W, Darzynkiewicz Z. Histone H2AX phosphorylation after cell irradiation with UV-B: Relationship to cell cycle phase and induction of apoptosis. Cell Cycle 4: 339-345, 2005;
Huang, X., Halicka, H.D., Traganos, F., Tanaka, T., Kurose, A., and Darzynkiewicz, Z. Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis. Cell Proliferat, 38: 223-243, 2005.)