[19 Chromosome Sorting by Flow Cytometry

By Marty Bartholdi, Julie Meyne, Kevin Albright, Mary Luedemann, Evelyn Campbell, Douglas Chritton, Larry L. Deaven, and L. Scott Cram

The 24 human chromosome types can be sorted by flow cytometry with 90% purity in quantities sufficient for chromosome specific DNA library construction1 or direct hybridization on filters.2 The techniques that need to be brought together to sort one million chromosomes of a single type, or 50 thousand of each type, on a commercially available flow cytometer are cell culture, large-scale chromosome preparation, resolution of single chromosome types, and long-term sorting at optimum rates and purity.

The cell types most commonly used as sources of chromosomes for sorting are diploid human fibroblasts, lymphoblasts, and Chinese hamster-human hybrids retaining one or a few human chromosomes. Human chromosomes purified from hybrid cells by flow cytometry have an advantage over the direct use of hybrids because the cross species background is removed. Chromosome sorting by flow cytometry can also be applied to cell strains with abnormal chromosomes or those with important phenotypic traits.

Flow cytometers operate on the principle of rapid analysis of single cells or chromosomes. A suspension of fluorescently stained chromosomes flows at a rate between 1000 to 2000/sec through a finely focused laser beam. Individual chromosome types are resolved on the basis of DNA content and base composition as determined by the cytochemistry of the fluorescent dyes. The chromosomes flow in a small stream that is broken

1 K. E. Davies, B. D. Young, R. G. Ellis, M. E. Hill, and R. Williamson, Nature (London) 293, 376(1981).

2 R. V. Lebo, D. R. Tolan, B. D. Bruce, M. C. Cheung, and Y. W. Kan, Cytometry 6, 478 (1985).

Copyright © 1987 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 151 All rights of reproduction in any form reserved.

into uniform droplets. The droplet containing the desired chromosome type, as indicated by the fluorescence measurement, can be deflected from the mainstream. Optimal sorting rates are 40 to 50 chromosomes/sec. Sorting can be done separately in two directions for production of one million copies of each of two chromosome types in 6 hr.

Purity is determined by the resolution of the desired chromosome type, the fraction of chromosome debris in the preparation, and instrumental factors. Purity is judged to be near 90% and preliminary characterization of flow sorted chromosomes confirms this. Both cytogenetic analysis and hybridization of chromosome specific probes to flow sorted chromosomes indicate that contamination is about 10%.3

In addition to the operation of the flow cytometer, the culturing of cells and chromosome preparation techniques must be highly efficient. A chromosome preparation should provide uniformly stained chromosomes, low debris levels, a high yield of free chromosomes from mitotic cells, and for library construction, chromosomal DNA of very high molecular weight. The yield of free chromosomes is crucial because low yields decrease the chromosome number concentration while increasing the debris fraction, making the chromosome sorting rates much slower than optimum. Techniques for chromosome preparation, resolution of the normal human chromosomes for flow sorting, and the way these interact to produce sorted chromosomes of high purity at optimum rates are described.

Chromosome Preparation

We have routinely prepared chromosomes from diploid human fibroblasts (foreskin strain HSF-7 developed by D. Chen, Los Alamos), diploid human lymphoblasts (GM 130A, Mutant Cell Repository, Camden, NJ), and a number of Chinese hamster-human hybrids. Each cell strain has its own characteristics that requires adjustments to each step in the preparation protocol. The basic steps in chromosome preparation are ( 1 ) culture of a sufficient number of cells, (2) blocking cells at metaphase with colcemid and harvest of mitotic cells, (3) cell swelling in hypotonic buffer, (4) dispersal of chromosomes in the presence of isolation buffer for stabilization of free chromosomes, and (5) staining of chromosomes with DNA specific dyes, Hoechst 33258 and chromomycin A3.

Cell Culture

Chromosome preparation for flow sorting requires from 10 to 100 times the number of mitotics considered necessary to produce the desired

3 B. Moyzis (Los Alamos National Laboratory), personal communication.

number of sorted chromosomes because the yield of isolated chromosomes is usually between 1 and 10% of the expected number. For a laige scale chromosome preparation from cells grown in monolayer (fibroblasts or somatic cell hybrids), 10 T150 flasks are seeded with approximately one million cells each. The culture media is a minimal essential media plus 10% fetal calf serum and the cells are grown at 37° in a 5% C02 incubator. Monolayer cells are maintained in exponential growth for at least two generations prior to starting the mitotic block. Beyond a certain passage number (about 20, but different for each culture) most human cell strains have an increasing number of senescent cells and become unsatisfactory for high quality chromosome preparations.

For chromosome preparation from lymphoblasts grown in suspension culture, four T75 flasks are seeded with 16 million cells each at a concentration of 3-4 X 105 cells/ml. The cell culture media is RPMI plus 10% fetal calf serum, and the flasks are placed in 5% C02 incubators at 37°. A single generation time is allowed prior to mitotic arrest. The extent of cell culture required for lymphoblasts is significantly less than that for monolayer cells. Also, chromosome yields of 10% and higher are routine, and yields of 20% have been obtained.

Mitotic Arrest and Harvest

Colcemid (0.1 //g/ml) is added to exponentially growing monolayer or suspension cell cultures. The optimal duration of the mitotic block varies considerably for each cell culture, but is typically 12 to 14 hr for diploid human fibroblasts, lymphoblasts, and hamster-human hybrids. Mitotic blocks of shorter duration do not allow as many cells to reach metaphase, and result in preparations of high resolution, low debris, but low chromosome number concentration. Prolonged Colcemid blocks increase debris levels by formation of micronuclei and colcemid toxicity. Increasing the colcemid concentration to increase disruption of the metaphase has been unsuccessful.

Mitotic cells are selectively detached from monolayer cultures by shake off. Usually hybrid mitotic cells are attached less firmly than human fibroblasts and require less vigorous shaking. The flasks are rapped with the palm and examined for the number of cells floating and attached. About 5 to 7 million cells can be harvested from each of the 10 flasks, and the fraction of mitotic cells in the harvest should be between 50 and 95%.

Because the mitotic index of a lymphoblast suspension culture is typically 30%, of the 20 million cells harvested from each of the four flasks seeded, about 6 million are mitotic cells. The mitotic index in the suspension, or fraction of mitotics harvested by shake off, can be determined by swelling cells in 1.0 ml hypotonic sucrose (34 g/liter) after centrifugation of an 8-ml sample of cell culture for 5 min at room temperature. The sample is again centrifuged and suspended in 5 ml of methanol, acetic acid (3:1) fix. Mitotic and interphase cells can then be scored under the microscope after staining with propidium iodide.

Cell Swelling

The harvested mitotic cells are allowed to swell in hypotonic buffer to loosen the metaphase chromosomes but not to the point of cell lysis. Careful modification of the hypotonic buffer used, and duration of cell swelling time, can significantly improve the yield of free chromosomes. The failure of the cells to swell uniformly or premature lysis are major factors in the loss of yield.

The harvested cells are pooled and counted in a Coulter counter to determine the number concentration. The cells are distributed at about six million mitotic cells each in 50-ml centrifuge tubes (account for mitotic fraction). In a large-scale preparation of fibroblasts or hybrids, 10 tubes are produced and for lymphoblasts, 4 tubes. The cell harvest is centrifuged (250 g for 10 min) and the cell culture media is aspirated. Fibroblasts or hybrid cells are resuspended in 5.0 ml, 75 mM KC1 for 25 min at room temperature. Lymphoblasts are resuspended in 55 mM Ohnuki's buffer (equimolar solution of KC1, NaN03, and NaC2H302 in a ratio of 10:5:2) for 60 to 100 min, but good yields have also been obtained using 75 mM KC1 for 20 min. Leaving the cells too briefly in hypotonic buffer does not swell the cells sufficiently. Prolonged swelling may lyse cells and release chromosomes (usually in clumps) prematurely. A second centrifugation and resuspension in a "wash" buffer (see below) sometimes improves the resolution and lowers debris levels, but can reduce yield and therefore is not routinely done in large-scale preparations.

Chromosome Dispersal

The swollen mitotic cells are centrifuged and resuspended in isolation buffer for physical dispersal of the metaphase chromosomes. Three isolation buffers have been used to stabilize the morphology of the free chromosomes in suspension. The final concentrations of the three buffers are listed here. Enzyme grade reagents are used, when available.

Buffer I (hexylene glycol),* final concentrations: 750 mM hexylene glycol

4 W. Wray and E. Stubblefield, Exp. Cell Res. 59, 469 (1970).

25 mM Tris base

1 mM MgCl2 0.5 mM CaCl2

Adjust pH to 7.5 using HC1 prior to addition of hexylene glycol, filter through 0.2-jum filter, store cold or freeze. This buffer can be prepared concentrated and diluted just prior to use. Buffer II (MgSOJ,5 final concentrations: 9.8 mM MgS04 48 mM KC1

4.8 mM HEPES

2.9 mM dithiothreitol

For 10 ml buffer add 1.0 ml of 100 mM MgS04, 9 ml of 55 mM KC1, and 5.5 mM HEPES, and 0.25 ml dithiothreitol (120 mM in H20), filter through 0.2-jum filter. Adjust pH to 8.0.

Buffer III (polyamine),6'1 final concentrations: 15 mM Tris base

2 mM EDTA 0.5 mMEGTA 80 mM KC1 20 mM NaCl

0.2 mM spermine 0.5 mM spermidine 14 mM /?-mercaptoethanol 0.1% digitonin (approximately)

Each of the first five components are prepared separately at 10 times the concentration given above, filtered through a 0.2-nm filter, and stored at room temperature. Spermine and spermidine stocks are prepared in distilled water at 0.4 and 1.0 M, respectively, and frozen in 50 jul aliquots. The isolation buffer is prepared on the day of use by combining 10 ml each of the first five stock buffer components (10X) plus 50 ml of distilled H20. The pH is adjusted to 7.2 with HC1 or NaOH. /?-Mercaptoethanol is added to a final concentration of 14 mM. (The buffer as prepared to this point can be used as a chromosome "wash" buffer.) To 25 ml of the wash buffer 30 mg of digitonin is added and the solution heated at 37° for 45 min. The undissolved digitonin is removed by filtration. Because not all of the digitonin goes into solution, the final concentration is not 0.12% but probably closer to 0.1%. Add 12.5 /ul of each stock solution of spermine and spermidine. Keep the chromosome isolation buffer cold.

5 G. van den Engh, B. Trask, L. S. Cram, and M. Bartholdi, Cytometry 5, 108 (1984).

6 A. B. Blumenthal, J. O. Dieden, L. N. Kapp, and J. W. Sedat, J. Cell Biol. 81, 255 (1979).

7 R. Sillar and B. D. Young, J. Histochem. Cytochem. 29, 74 (1981).

When dispersing chromosomes in Buffer I (hexylene glycol), the mitotic cells from the cell swelling step are centrifuged and resuspended in 0.5 ml buffer and incubated at 37° for 10 to 20 min and then placed on ice for 60 min. The chromosomes are dispersed by forcing the swollen cells through a 1.5 in., 22-gauge syringe needle. The needle is bent slightly to allow the beveled tip to lie flat against the side of the centrifuge tube wall and the syringed suspension should fan out down the wall with little foaming. The suspension is syringed one to three times and the chromosomes are monitored after each pass. Undersyringing results in undispersed chromosomes and oversyringing in chromosome fragmentation, both with significant loss of yield. Usually both conditions are present together and the optimal number of passes must be evaluated by flow cytometry. While microscopic observation is important at this step, it is not a reliable predictor of flow karyotype quality.

Chromosomes from human fibroblasts prepared in Buffer I retain their morphology and can be banded after sorting. This method has been used to confirm the chromosome identity of the peaks in the flow histogram. Purity can also be judged during sorting by identifying each of the sorted chromosomes to determine the number of contaminants. Variable quality of banding, however, makes this practice difficult to implement routinely. The major disadvantage of Buffer I has been high levels of debris, particularly under the smaller chromosomes.

When using Buffer II (MgS04), the mitotic cells in each tube are resuspended in 1.0 ml buffer, and allowed to stand for 10 min at room temperature. The detergent Triton X-100 is then added to a final concentration of 0.25% for fibroblasts and 1% for hybrids. The suspension stands for an additional 10 min before syringing as described above. Chromosomes from human fibroblasts and hamster-human hybrids prepared in Buffer II usually have excellent uniformity of dye staining, moderate debris levels, and good yields.5 A disadvantage is difficulty in identification of sorted chromosomes due to a high degree of condensation.

In our laboratory the most consistent results have been obtained with Buffer III (polyamine). The polyamines condense the chromosomes rendering identification difficult but banding has been reported.2 The resolution, debris levels, and yields are usually superior, especially with the lymphoblast cell line GM 130A.

For preparation of chromosomes in Buffer III, after cell swelling in hypotonic buffer and centrifugation, the cell pellet is resuspended in 1.0 ml cold polyamine isolation buffer. The chromosomes are then dispersed by placing the centrifuge tube on a vortex mixer for up to 60 sec. The time should be adjusted for each cell type. After 60 sec, if more vortexing is indicated, cool the tube before continuing. The presence of chelating agents and cold inhibits nuclease activity, and dispersal by vortexing in the presence of detergent gives more uniform yields.7 A small portion (10 ^1) of the chromosome preparation can be stained with an equal volume of propidium iodide (50 //g/ml) and examined microscopically. An acceptable preparation contains mostly free chromosomes with few clumps or fragments.

Chromosome preparations in Buffer III (polyamine) can be stored unstained at 4° and have long shelf life. Some preparations have been stored for over 18 months with no loss of resolution or chromosomes. Preparations in Buffer I (hexylene glycol) are stored frozen, unstained, and preparations in Buffer II (MgS04) are stained immediately and should be used within 1 week.

A typical chromosome preparation from fibroblasts and hybrids will produce 10 tubes containing 1.2 ml each with a free chromosome number concentration of about 2.5 million/ml. This corresponds to 3 million chromosomes per tube in which about six million mitotics were swollen for a yield of 1%. About 50% of the preparation is chromosome debris. From the 10 tubes, about one million copies of a single chromosome type can be sorted. Two million chromosomes, one million of two chromosome types, can be sorted with two way sorting.

An optimal preparation from diploid lymphoblasts produces four tubes containing 1.2 ml each and a concentration of free chromosomes of 25 million/ml. This corresponds to 30 million chromosomes per tube or a yield of 10%. Yields of up to 20% have been obtained. The debris is mostly interphase nuclei that can be removed by slow speed centrifugation (75 g, 3 min). Typically, 4 million chromosomes of a single type can be sorted from the total preparation of 4 tubes (two types for two way sorting).

Each step in the isolation protocol, especially metaphase block time, hypotonic treatment, and optimum mitotic cell number concentration, must be adjusted for each cell strain and the results evaluated by flow cytometry. Improvements in yields while maintaining chromosome integrity are needed. The major factor affecting yield appears to be the cell type, with the lymphoblasts routinely more productive than fibroblasts, and with some hybrids working better than others. Also, even with the same cell strain, the yield may be variable from preparation to preparation.

Chromosome Staining

The dyes, Hoechst 33258 and chromomycin A3, provide the optimum resolution of human chromosomes from diploid human cells and Chinese hamster-human hybrids.8 Hoescht 33258 binds to AT base pairs and

8 R. G. Langlois, L. C. Yu, J. W. Gray, and A. V. Carrano, Proc. Natl. Acad. Sci. U.S.A. 79, 7876(1982).

chromomycin A3 to GC base pairs. The chromosome types are resolved by relative DNA content and base composition.

The stock staining solutions are chromomycin A3, 0.5 mg/ml in Mc-Ilvane's buffer pH 7, 5 mM MgCl2 (1:1), and, Hoescht 33258, 0.5 mg/ml in distilled H20. Add 300 /¿I stock solution of chromomycin A3 to a 1.0 ml chromosome sample prepared in Buffer III at least 3 hr prior to sorting to allow the stain to reach equilibrium (final concentration, 125 fiM) and add 11.2 /¿I stock solution of Hoescht 33258 (final concentration, 4.8 fiM) just before sorting. The chromosome preparations degrade quickly after staining and should be used within 1 week. The stain concentrations given here are optimal for analysis on the EPICS V flow cytometer. For chromosomes prepared in Buffers I and II, lower stain concentrations of Hoescht 33258 (3 fiM) and chromomycin A3 (30 fiM) may be used. Uniformity of staining is excellent with chromosome buffers II (MgS04) and III (polyamine).

Chromosome Sorting

Chromosome sorting with high productivity and high purity requires clear resolution of the chromosome type, fast sorting rates, high yields, and low debris levels in the chromosome preparations. The characteristics of the preparations such as chromosome number concentration, uniformity of staining, and debris levels interact in a complex manner with the capabilities of the flow cytometer. The technique to be described applies to a commercial flow cytometer (EPICS V, Coulter Electronics, Inc., Hialeah, FL).

Resolution

The clear resolution of individual chromosome types gives flow cytometry the capability to separate chromosomes with high purity. Resolution depends partly on the intrinsic properties of the chromosomes and the cytochemistry of staining required to distinguish such differences in size and composition, and partly on the capability of the flow cytometer to measure small differences in fluorescence intensity among the chromosomes types with high precision. Resolution of chromosomes stained with Hoechst 33258 and chromomycin A3 is done with two argon ion lasers. Hoechst is excited in the ultraviolet (UV) and chromomycin A3 at 457.9 nm. Laser powers of 200 mW in the UV and 350 mW at 457.9 nm are sufficient for good precision, but lower powers should be used if feasible to prolong laser tube life (Innova 90-6 argon ion lasers, Coherent Inc., Palo Alto, CA). The two laser beams are focused to two separate illumination spots of 16 by 40 fim elliptical cross sections by a single pair of crossed cylindrical lenses of 80 and 40 mm focal lengths (confocal lens assembly).

Fluorescence measurements of higher precision may be possible with higher illumination intensity, but this requires special optics.2,9

The sample stream containing the chromosomes is hydrodynamically focused by an outer concentric sheath stream to a diameter of 3 //m. The focusing occurs within the 76 //m orifice of the flow nozzle. The square quartz nozzle (Coulter Electronics, Inc.) is easier to sort with routinely than stream-in-air because the process of breaking the stream into droplets sometimes perturbs the outer surface of the flow stream at the laser intersection. The illumination spot is aligned with its long dimension horizontal and the sample stream flows vertically through its center to provide uniform illumination to each chromosome.

The only special feature used in the EPICS V for chromosome analysis is the measurement of the two fluorescence signals on a single photomulti-plier tube. Because the two illumination spots are separate, the two signals occur sequentially in time. A pair of optical filters, KV408 (placed closest to the flow stream) and GG495 block the stray illumination light and light scatter from the chromosomes at the UV and 457.9 nm wavelengths. The fluorescence signal produced by UV excitation is proportional to Hoechst binding. It consists of the red edge of Hoechst fluorescence and chromo-mycin A3 fluorescence excited through energy transfer. Because energy transfer acts over a short range, only chromomycin A3 molecules adjacent to Hoechst molecules are excited to fluoresce by this mechanism. The fluorescence signal produced by 457.9 nm excitation is proportional to the total chromomycin A3 binding.

The two signals are processed with the electronics provided with EPICS V (Fig. 1). The fluorescence signals occur sequentially at the output of the photomultiplier tube and are integrated to provide a pulse that will produce a single window in the gated amplifier module. The sequential pulses are sent to two signal inputs of the gated amplifier module where they are separated. One set of signals is delayed to align the UV pulse in the gate window and the other set is undelayed to align the 457.9 nm pulse in the window. The action of the window serves to eliminate the pulse outside it. The fluorescence signals now can be amplified and integrated separately and are acquired in a bivariate histogram with 128 channels on each axis.

The fluorescence signals from Hoechst 33258 and chromomycin A3 stained chromosomes must be within a factor of 10 in relative intensities to be measured with a single photomultiplier tube. The stain concentrations and laser powers can be adjusted to accomplish this. A bivariate histogram of HSF-7 fibroblast chromosomes prepared in Buffer III (polyamine) and stained with Hoechst 33258 and chromomycin A3 is shown in Fig. 2. Each

9 M. Bartholdi, D. Sinclair, and L. S. Cram, Cytometry 3, 395 (1983).

Chinese Hamster Ovary ChromosomesChinese Hamster Ovary Chromosomes
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