The LLNL high‐speed sorter: Design features, operational characteristics, and biological utility

Abstract

This report describes a dual-beam, highspeed sorter (Hiss) with a droplet production rate of 220,000 s-l now in routine use at the Lawrence Livermore National Laboratory. The system can process and sort objects at rates in excess of 20,000 s-'. We report here on Key terms: High-speed sorting, dual-beam the development of HISS, describe its opera-flow cytometry, chromosome sorting, cell tional characteristics, and evaluate its utility sorting.for analysis and purification of human chromosomes, human erythrocytes, and living Chinese hamster ovary cells.The flow cytometer/cell sorter has become an important tool for the biomedical sciences, allowing identification and purification of subpopulations of cells and organelles with unequalled accuracy. During the last 15 yr cell sorters have been applied to purification of a broad range of cell and organelle types: for example, leukocyte and bone marrow subpopulations distinguished by their differential affinity for antibodies (8); human chromosomes distinguished by their differential uptake of one or more DNA-specific dyes (2); rare mutant erythrocytes and other peripheral blood cells distinguished by their altered affinity for antibodies against variant proteins (1); and malignant cells distinguished by their altered ability to scatter light and bind DNAspecific stains andor antibodies (11). Uses for sorted objects include simple visual study, biochemical analysis, molecular cloning, and functional assessment. Most commercially available cell sorters can scan several thousand cells per second, selecting from these the cells to be purified. Unfortunately, this processing rate is not adequate for many studies. For example, by processing objects at 2,000 s-', over 120 h of sorting time would be required to purify 1 pg of DNA from the human Y chromosome (3) and almost 140 h of sorting time would be required to sort 100 mutant erythrocytes from a population in which the mutant cell frequency wasWe have completed the development of, and put into routine operation, a dual-beam high-speed sorter (HISS) that reduces many of the limitations imposed by the relatively slow processing speed of conventional sorters. This system operates over ten times faster than conventional systems and is capable of processing and sorting cells at rates well in excess of 20,000 s-'.Most dual-beam flow cytometerhell sorters, including HiSS, operate according to the same principles (4,5,14,17). Briefly, cells or organelles in monodisperse suspension and stained with two or more fluorescent dyes are carried in single file in a fast-moving liquid stream sequentially through two tightly focused laser beams whose wavelengths are adjusted to excite the fluorescent dyes. The fluorescence andor light scatter produced during each laser beam crossing is collected by a n optical system and projected through spectral filters onto photodetectors. These detectors and their associated electronics convert the fluorescence andor scattered light to electrical pulses whose amplitudes are 'Work performed under the ...

Sorting of chromosomes by magnetic separation

“…In contrast, even high-speed flow sorters (Peters et al 1985) sort specific chromosomes at a rate not higher than about 1000 chromosomes/s, which corresponds to a few micrograms of chromosomes per hour. To realize the large sorting potential of the magnetic beads separation technique, it will be necessary, however, to overcome the severe clumping observed when larger numbers of chromosomes/ magnetic beads are used (data not shown).…”

Characterization and organization of DNA sequences adjacent to the human telomere associated repeat (TTAGGG)n

“…DNA sources Dual-beam high-speed sorting (18) Plasmid DNA preparation The alkaline lysis protocol for rapid plasmid DNA preparation was used as described (21).…”

Cytometry and Cytometers: Development and Growth

“…Los Alamos provided the inoculum for the subsequent growth of another major center for flow cytometer development at Lawrence Livermore Laboratory (Livermore, CA, USA), where high-speed flow sorting was perfected as a means for separating human chromosomes stained with a combination of A-T selective (Hoechst 33342) and G-C selective (chromomycin A 3 ) DNA dyes (Gray et al 1987;Peters et al 1985). The MoFlo high-speed sorter developed by Ger van den Engh and others at Livermore was subsequently refined by Cytomation (now DakoCytomation, Fort Collins, CO, USA), and has been produced commercially by them since 1994.…”