In situ hybridization to chromosomes stabilized in gel microdrops

Abstract

Conventional chromosome in situ hybridization procedures rely on fixation to glass slides followed by microscopic evaluation. This report describes the development of a microdrop in situ hybridization (MISH) method which facilitates hybridization to chromosomes in suspension. Chromosomes encapsulated in gel microdrops (GMDs) composed of an agarose matrix withstood stringent hybridization and denaturation conditions. Because of the increased stability, hybridization to encapsulated chromosomes was detected by flow cytometry as well as conventional microscopy. Thus, the MISH method offers a means for chromosome hybridization without slides and may enable identification and isolation of chromosomes using hybridization rather than nucleic acid binding dyes. Key terms: GMD, flow cytometry, MISHGel microdrop (GMD) technology (33) evolved from an interest in encapsulating biological materials such as mammalian cells or microorganisms in agarose microspheres. Growth (25,34), secretion (25,26,34), metabolism (34,35,36), cytotoxicity (2,15,35), and electroporation (14) can be measured rapidly in the defined microdrop environment. GMDs are usually composed of agarose. They are prepared by dispersing molten agarose into an excess of a hydrophobic fluid, such as inert silicone oil, to form an emulsion. After the emulsion is transiently cooled, GMDs are separated from the silicone oil by centrifugation and remain physically distinct and robust.Various methods are available to analyze metaphase chromosomes, including flow cytometry (7,17,2 1 ), in situ hybridization (16,19,29,30,31), and staining (3,10). In order to provide stability, chromosomes are typically adsorbed onto slides for analysis. Consequently, analysis requires microscopic evaluation of individual slides which limits automation. Attempts to perform in situ hybridization on chromosomes in solution have been hindered by clumping, breakage, and aggregation ( 1 ). Current methods ( 18) could be improved by increased stabilization. Integration of GMD technology with hybridization techniques provides a system for analyzing chromosomes in suspension, facilitating sample handling, flow cytometry analysis, and hybridization-based isolation.GMDs provide assay microenvironments which are compatible with most in vitro cell manipulations. Our studies have indicated that GMDs can be pipetted, centrifuged, filtered, and analyzed by flow cytometry. The gel matrix provides physical protection, while the small size and porosity result in high permeability and rapid diffusion (35). These properties make the GMDs compatible with enzyme manipulation, fluorescence staining, and repeated washing.The results presented here demonstrate successful stabil ization and flow cytometric analysis of hybridized chromosomes. Development of this technology involved optimization of buffers, emulsion speeds, surfactants and agarose type. Successful encapsulation of chromosomes into GMDs was first confirmed using fluorescence microscopy and image analysis. Protocols were then developed for in situ ...

Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses.

“…(A related strategy, based again on restricting the volume containing the determinand to be analyzed, has in fact permitted the fluorimetric detection and resolution of individual fluorescent molecules [676], while the [nonflow] cytometric assessment of ␤-galactosidase activity in single bacterial cells was performed more than 30 years ago [822].) In the studies by Nir et al (684), the beads were used to isolate yeast mutants growing at different rates, while the Massachusetts Institute of Technology group have analyzed antibody secretion by hybridoma cells (775), drug-resistant subpopulations and the nutrient sensitivity of hybridoma cells (1011), and chromosome hybridization (677). Tuberculosis is a reemergent infection of great concern (94,99,827,923,1056), and its analysis and control are greatly hampered by the time necessary (as a result of the slow growth of pathogenic mycobacteria) for identification and susceptibility testing.…”

Single-Cell Sorting of Microorganisms by Flow or Slide-Based (Including Laser Scanning) Cytometry

“…Specific DNA and RNA sequences in the nuclei are detectable by FISH and dual-laser FCM; the combined method is sometimes so sensitive that single HeLa cells can be analyzed for a particular RNA [37]. Chromosomes can be made stable before hybridization by being allowed to dry enough on microscope slides for adherence [1], or they can be stabilized in GMDs [38]. The amounts of specific sequences of DNA were estimated by measurement of fluorescent-spot intensities from FISH combined with digital imaging microscopy [39].…”

Single-Cell Sorting of Microorganisms by Flow or Slide-Based (Including Laser Scanning) Cytometry

“…Specific DNA and RNA sequences in the nuclei are detectable by FISH and dual-laser FCM; the combined method is sometimes so sensitive that single HeLa cells can be analyzed for a particular RNA [37]. Chromosomes can be made stable before hybridization by being allowed to dry enough on microscope slides for adherence [1], or they can be stabilized in GMDs [38]. The amounts of specific sequences of DNA were estimated by measurement of fluorescent-spot intensities from FISH combined with digital imaging microscopy [39].…”