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Titolo:
"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: Potential application at microgravity conditions in space
Autore:
Smolewski, P; Bedner, E; Gorczyca, W; Darzynkiewicz, Z;
Indirizzi:
New York Med Coll, Brander Canc Res Inst, Hawthorne, NY 10532 USA New YorkMed Coll Hawthorne NY USA 10532 es Inst, Hawthorne, NY 10532 USA New York Med Coll, Dept Pathol, Hawthorne, NY 10532 USA New York Med CollHawthorne NY USA 10532 Pathol, Hawthorne, NY 10532 USA Med Univ Lodz, Dept Hematol, Lodz, Poland Med Univ Lodz Lodz PolandMed Univ Lodz, Dept Hematol, Lodz, Poland Pomeranian Sch Med, Dept Pathol, Szczecin, Poland Pomeranian Sch Med Szczecin Poland h Med, Dept Pathol, Szczecin, Poland
Titolo Testata:
CYTOMETRY
fascicolo: 4, volume: 44, anno: 2001,
pagine: 355 - 360
SICI:
0196-4763(20010801)44:4<355:"CSBDD>2.0.ZU;2-U
Fonte:
ISI
Lingua:
ENG
Soggetto:
APOPTOSIS;
Keywords:
laser scanning cytometry; DNA staining; protein staining; DAPI; propidium; sulforhodamine 101;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
17
Recensione:
Indirizzi per estratti:
Indirizzo: Darzynkiewicz, Z New York Med Coll, Brander Canc Res Inst, 19 Bradhurst Ave,Suite 2400, Hawthorne, NY 10532 USA New York Med Coll 19 Bradhurst Ave,Suite 2400 Hawthorne NY USA 10532
Citazione:
P. Smolewski et al., ""Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: Potential application at microgravity conditions in space", CYTOMETRY, 44(4), 2001, pp. 355-360

Abstract

Background: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinsesmay be burdensome in certain situations such as field expeditions or combat. Methods: The "liquidless" staining procedure is proposed in which the dyesare contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm(2)) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensityof fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). Results: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was foundto be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristiccellular DNA content frequency histograms with distinct G(1), S, and G(2)/M cell populations and 2:1 ratio of G(2)/M to G(1) peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. Conclusions: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The qualityof the staining provided by this methodology is comparable to conventionalcell staining in dye solutions. Cytometry 44:355-360, 2001. (C) 2001 Wiley-Liss, Inc.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 04/12/20 alle ore 00:50:54