Counterstaining human chromosomes for flow karyology

Abstract

For example, when human metaphase chromosomes stained with the AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI), are counterstained with the nonfluorescent AT-specific DNA-binding agent, netropsin (NTR), the G-banding-like pattern of the DAPI fluorescence along the chromosome arms is depressed. The fluorescence of heterochromatic C-band regions of chromosomes 1,9,15,16, and Y is not depressed, however, and remains bright (12). Use of NTR as a counterstain with the GC-specific fluorochrome chromomycin A3 (CA3) increases contrast of the R-banding-like pattern of CA3 (41. With a triple-stain combination of CA3, NTR, and DAPI, one can observe the R-banding pattern of CAB by exciting at 458 nm and the regional DAPUNTR banding pattern by exciting in the ultraviolet (UV) (9).We have adapted this counterstaining technique with DAPI, CA3, and NTR for bivariate flow karyology on a dual-laser EPICS V flow cytometer. Human metaphase chromosomes were isolated in a manner similar to that developed for Chinese hamster chromosomes, and the three stains were added together in a buffered medium (14). Flow cytometric analysis was done on an unmodified EPICS V flow cytometer equipped with two (5-W all-lines visible) argon-ion lasers operating in the UV and 458 nm.While the number of individual chromosome types resolved was less than that resolved by univariate analysis of propidium iodide (PI) stained chromosomes analyzed on an experimental flow cytometer, or in bivariate analysis of Hoechst 33258 (HO) and CAB stained chromosomes analyzed on the EPICS V flow cytometer, the discrimination of the 1,9, and Y chromosomes was striking, as was the resolution of variations detected in chromosome 9 homologues. These results indicate that a fluorescent banding technique developed for microscopy can be adapted to flow cytometry and that the resolution of individual chromosomes and subchromosomal regions can facilitate flow karyology and chromosome sorting. MATERIALS AND METHODS Chromosome Preparation and StainingChromosomes were isolated from human foreskin fibroblast cell strains using the method of Van den Engh et al. (14). Cells were blocked in mitosis with colcemid (3 x 10W7M) for 5 h. Mitotic cells were harvested by shaking off, centrifuged at 150 g for 8 min, and the supernatant was removed. One milliliter of swelling

Chromosome banding analysis by slit‐scan flow cytometry

“…We have shown previously that chromosomes can be banded in suspension by use of a counterstain and can be resolved by a total fluorescence measurement (20). Here, the total fluorescence measurement was preserved after induction of low contrast banding by chromomycin A3 to chromosomes in suspension.…”

“…After swelling, the cells were centrifuged and resuspended in the chromosome isolation buffer (15 mM Trishydrochloride, 2 mM EDTA, 0.5 mM EGTA, 80 mM KC1, 20 mM NaC1, 14 mM beta-mercaptoethanol, 0.2 mM spermine, 0.5 mM spermidine, and 0.1% digitonin). The chromosomes were dispersed by vortexing at high speed for 30 s and stained with 125 pM chromomycin AS.…”

“…The 24 human chromosomes are usually resolved into 21 distinct peaks with the 9-12 chromosomes falling in a single peak by this technique (2). These four chromosomes can now be resolved by special techniques, such as counterstaining (20) or by the use of hamster-human hybrid cells (3).…”

Chromosome Analysis Using Benchtop Flow Analysers and High Speed Cell Sorters

“…Similar chromosome resolution was also acquired from the sorter BD Influx using this dye combination albeit at a much higher laser powers (100 mW of 355 nm, 200 m of 488 nm). We speculate that this binding phenomenon could either be due to the spectral energy transfer or binding competition which occurs between the pair of DNA dyes used . However, the chemistry behind the chromosomal binding properties of PI with other DNA dyes is beyond the scope of this article.…”

Overview of flow cytogenetics for clinical applications

“…Later, fluorochromes with nucleotide (A:T or G:C) specificity provided the means for resolving most of the human karyotypes (Langlois et al, 1982). Other techniques have been developed that stain heterochromatin (Meyne et al, 1984;Lebo et al, 1984) and label polynucleotide sequences.…”