Cultivation of Cells

The authors have developed a method for large-scale isolation of metaphase chromosomes from HeLa cells. The distinguishing feature of this method is the use of a pH sufficiently low (about 3) to stabilize the chromosomes against mechanical damage. Many milligrams of fairly pure, morphologically intact chromosomes can be isolated in 8 hr or less of total working time. The isolated chromosomes contain about 2.0 mg of acid-soluble protein, 2.7 mg of acid-insoluble protein and 0.66 mg of R N A for each milligram of DNA. The R N A bound to the isolated chromosomes consists mainly of ribosomal RNA, but there is also a significant amount of 45S RNA. I N T R O D U C T I O N Many possible biochemical and biophysical approaches to the study of chromosomes in higher organisms have been hindered, until recently, by the lack of suitable procedures for large-scale isolation of chromosomes. Although the methods for isolation of interphase chromosomes, or "chromatin," which have been developed in recent years (1, 2) are satisfactory for certain purposes, a definite need still exists for a procedure which will allow large-scale isolation of morphologically intact metaphase chromosomes. Metaphase chromosomes are an indispensable complement to interphase chromosomes for the general study of chromosome structure. In addition, metaphase chromosomes have the unique advantage of being so condensed that they can be distinguished microscopically both from each other and from contaminating nonchromosomal material. Consequently, one is not limited to studying the average properties of all chromosomes; one can also examine single types of chromosomes. According to our experience, in the isolation of metaphase chromosomes by most previously published methods (3-5), morphological damage to some of the chromosomes cannot be avoided and only partial purification of the chromosomes from cell debris can be achieved. We report here a method for the rapid preparation, in milligram quantities, of fairly pure, morphologically intact metaphase chromosomes from HeLa cells. We also report the results of studies on the chemical composition of isolated chromosomes. M A T E R I A L A N D M E T H O D S Cultivation of Cells HeLa $3 cells (6) were grown in suspension culture in a modified Eagle's medium (7) supplemented with 5% calf serum. For accumulation of metaphase cells, partial synchrony was induced by lowering the culture temperature to 4°C for 1 hr and then returning it to 37°C (8). Ten to 11 hr later, colchicine was added to a final concentration of 0.5 to 1 X 10 -5 M. The cells were harvested by centrifugation 9 to 10 hr after colchicine addition and washed 3 times in 0.137M NaC1, 0.005M KC1, 0.007M NaH2PO4, 0.025 m Tris, pH 7.4. This procedure routinely produced about 30% metaphase cells. 95 on July 4, 2017 jcb.rress.org D ow nladed fom Isolation of Chromosomes All operat ions were carried out in the cold (0 ° to 4°C). T h e pellet of washed cells was gent ly resuspended in 15 vol of 0.1M sucrose, 7 X 10-4M CaC12, 3 X 10-4M MgCI2 (4). T h e cells swelled in this hypotonic m e d i u m and the chromosomes in metaphase cells became excellently separated f rom each other. Five rain later, 3 vol of 0.1 M sucrose, 7 X 10-4M CaCl~, 3 X 10-4M MgC12, 3.3 X 10-aM HC1 were added slowly, with stirring, to each vo lume of cell suspension. Slow addi t ion of the acid solution was necessary to prevent c lumping of the chromosomes in me taphase cells. T h e measured final p H was about 3.0. Higher p H values (up to 3.3) allowed satisfactory breakage of cells and conservat ion of chromosome morphology, bu t separa t ion of the chromosomes from cytoplasmic debris was more difficult. A phase-contras t microscope was used to check the result of acid addition. Cells suspended in hypotonic m e d i u m appeared grey, with little internal contrast. T h e chromosomes in me taphase cells were barely visible. After the p H h a d been adjusted to 3.3-3.0, the chromosomes, evenly dis t r ibuted th roughout the cytoplasm of metaphase cells, appeared dist inct and bright. After ad jus tmen t of pH, a Pot ter -Elvehjem glass homogenizer with a motor -dr iven Tef lon pestle was used to homogenize the ceils. T h e course of homogeniza t ion was checked with a microscope. As a n end point for homogenizat ion, the t ime was chosen w h e n all in terphase ceils were broken (usually after less t h a n 1 rain). At this stage the great major i ty of me taphase cells were also broken. T h e released chromosomes were usual ly single and free of obvious a t tached debris. T h e following steps separated these chromosomes from the nuclei a n d cytoplasmic debris which were also produced by homogenizat ion. T h e homogena te was centr ifuged at 900 g (2000 RPM in the In te rna t iona l PR2 centrifuge, head No. 269, In te rna t iona l E q u i p m e n t Co., N e e d h a m Heights, Massachuset ts) for 30 min. The result ing pellet conta ined nuclei, chromosomes, and the larger cytoplasmic debris. Most debris r ema ined in the supernatant . T h e supe rna t an t was discarded and the pellet r e s u s p e n d e d i n H C M (1 X 10 -3 M HC1,7 X 10 -4M CaC12, 3 X 10 -4 M MgCI)2, us ing about 40 ml of H C M for each milliliter of pellet. T h e suspension was rehomogenized briefly with a Pot ter -Elvehjem homogenizer to break u p any c lumps tha t migh t have formed as a result of pelleting. U p to 20 ml of suspension at a t ime were then gent ly layered onto 200 nfi of a 0.1 to 0.8 M linear sucrose gradient in H C M (final p H adjusted to 3.0) which h a d been formed in a 250 ml glass centrifuge bottle. The gradient was accelerated at 500 RPM per min to 1500 RPM (450 g) in the In ternat ional PR-2 centrifuge, head No. 284, and held at tha t speed for 20 min. Deceleration was also at 500 RPM per nlin. After the centr i fugat ion the chromosomes were dist r ibuted from near the bo t tom of the gradient to near the top. Cytoplasmic debris r emained at or near the top, extending into the ch romosome region. Nuclei and some clustered chromosomes were pelleted at the bot tom. A crude fract ionat ion of chromosomes on the basis of sed imenta t ion velocity was also produced; most large chromosomes were found near the bot tom, while most small chromosomes r ema ined near the top. T h e top 20 ml of the grad ien t were discarded and the rest was sucked off, leaving a small a m o u n t (about 10 ml) in the bo t tom of the centrifuge bottle so as not to dis turb the pelleted nuclei. T h e superna t an t was then mixed unt i l the sucrose was evenly distributed, and the chromosomes were collected by centr i fugat ion at 850 g (2000 RPM ill the In ternat ional PR-2 centrifuge, head No. 284) for 90 min. T h e pellet contained very few nuclei (less t h a n 3% of the total D N A in the pellet was from whole nuclei if the initial propor t ion of me taphase cells was 15% or greater). The re was, however, still considerable cont amina t ion by debris. Mos t of the debris was removed by the following procedure. T h e pellet was resuspended in a small vo lume of H C M with brief rehomogeniza t ion to break up clumps. T e n ml of 2.2 M sucrose in H C M were placed in a Spinco SW-25 plastic tube (Beckm a n Ins t ruments , Inc., Palo Alto, California) and 15 to 20 ml of ch romosome suspension were layered on top. T h e upper three-fourths of the tube contents we re g e n t l y s t i r red to f o r m a r o u g h g r a d i e n t . Af te r centr i fugat ion at 20,000 RPM for 1 h r the ch romosomes were found in a pellet at the bo t tom of the tube, while most cytoplasmic debris r ema ined floating above the 2.2 M sucrose layer. T h e yield of chromosomes at this point, as de te rmined by D N A determ i n a t i o n (see be low) or by d i rec t c o u n t i n g in a Petroff-Hausser count ing chambe r (C. A. Hausser and Son, Phi ladelphia, Pennsylvania) , was about onethird of the chromosomes f rom all cells scored as in metaphase before homogenizat ion.