Preparing Metaphase Spreads: From Painful to Perfect (2023)

Cytogenetics is a branch of genetics dedicated to the study of chromosomes. Chromosome instability and chromosome rearrangements in the genome are used to diagnose and define several genetic diseases and even some cancers. [1, 2] Researchers use a variety of techniques to identify the structure of chromosomes, including fluorescence in situ hybridization (FISH), karyotyping,flow cytometry,and comparative genomic hybridization (CGH).

Even if you’re not a cytogeneticist, you might need to evaluate the chromosome instability in a certain cell line, for example, before you use them for your thesis project (especially if you are using embryonic stem cells). [3,4]

The first step in assessing chromosome instability or chromosome rearrangement is harvesting chromosomes, which can be a bit of an art form in itself.

First Steps in Preparing Metaphase Spreads

First, your cells need to be about 80% confluent (and healthy!) before you harvest them. If you don’t have enough cells, you may not end up with enough chromosomes for further analysis.

Arresting the Cell Cycle

The next step is to arrest the cell cycle. Arresting the cell cycle in metaphase is crucial for studying chromosomes because this is the point at which they are the most condensed, allowing for easier visualization. A few common metaphase arresting agents include colchicine, demecolcin, vinblastine, and nocodazole. [5,6]

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Demecolcin, or colcemid, is the most common compound used to arrest the cell cycle for metaphase spreads. [7,8] Colcemid is a synthetic analog of colchicine, which is highly toxic. [9,10] Vinblastine, like colchicine, is a natural compound found in plants but has also been shown to affect DNA synthesis, which is most likely why it is not used for metaphase spreads. [11,12]

All four of these compounds arrest the cells in metaphase by destabilizing microtubules, which prevents spindle assembly and activates the spindle assembly checkpoint. [5,6] The cells will not be able to proceed into anaphase when incubated with any of these compounds.

Time to Harvest

Once you have arrested your cells in metaphase, it’s time to start harvesting! This almost works the same way as harvesting cell pellets, except you’ll retain the media with the colcemid, rather than pouring it away. Pour the colcemid-spiked media into a conical tube, then trypsinize your cells. Once the cells are detached, dilute the trypsin with the saved colcemid-spiked media. Centrifuge your cells, remove the supernatant, and you’ll have a pellet of metaphase-arrested cells.

Here comes the first tricky part: making the cells fragile. For metaphase spreads, we need to rupture the cell membrane to help visualize the chromosomes.The cells need to be delicate so that they will burst when dropped onto a microscope slide.

To do this, we need to add a hypotonic solution to the cells, which has a lower concentration of solutes relative to the inside of the cell. This causes an osmotic gradient to form between the outside and the inside of the cells. Thus, adding the hypotonic solution causes the cells to swell, making them very fragile. Think of the cells like water balloons: you want them full enough so that they’ll burst when they hit something, but not full enough to break in your hands!

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The easiest way to achieve this balance is to add the hypotonic solution a little at a time, rather than the whole amount at once. This way, you can resuspend your cells slowly, allowing for uniform swelling. You want to make sure that there are no clumps of cells in your tube. Gently tapping on the tube should break up any unwanted cell clumps. The most common hypotonic solution for metaphase spreads is potassium chloride, but some protocols use sodium citrate. [13-15]

Hold That Thought

Once the cells have been incubated with the hypotonic solution, you want to fix them in this swollen state so they can be stored for future use. A combination of methanol and acetic acid seems to be the most common fixation method for chromosome harvesting. Using an alcohol, like methanol, alone will result in the cells shrinking. Adding the acetic acid, which is associated with swelling, preserves the cells in their not-too-swollen state. You can store these cells as a pellet at 4°C for a year or go directly to the next step. [7]

Preparing the Slides

Getting the metaphase chromosome spreads onto the slides is the most difficult part of chromosome harvesting, and there is plenty of debate on which step is the most influential on the final spread.

Once you are ready to make your metaphase spreads, resuspend your cell pellet in fresh fixative a few times. This will help get rid of any cell clumps before you drop them onto your slides.

The trick is to let gravity do the work for you, but you may need to spend some time optimizing the distance. If the distance between your slide and your pipette is too large, your chromosomes will spread too far apart. When chromosomes are too far apart, it may be difficult to determine if they were from one cell or two cells. This could be a huge problem if you are trying to assess the ploidy of your cells. [16]

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If the distance between your slide and your pipette is too small, your chromosomes will overlap and be squished together. When your chromosomes are overlapping, you won’t be able to tell them apart. This will make any staining you might do difficult to evaluate. Use a disposable transfer pipette to drop your cells onto clean slides, but keep in mind that the diameter of the pipette can also influence chromosome spread on the slide.

Humidity is a critical component of the metaphase spread procedure, and it seems to be the most important factor affecting how the chromosomes spread on the slides. [17] The fixative pulls moisture from the air, which results in rehydration of the now broken cells. Controlling this rehydration process is the key to beautiful metaphase spreads!

The two most common methods of maintaining slide humidity are:

  • keeping the slides slightly damp with water (either by dipping the slides in water or pre-freezing them) before dropping the cells onto them, then allowing them to dry on a heat block.
  • using dry slides, dropping the cells, and then immediately placing the slides on a tray in a covered water bath.

Choosing the method is up to the individual, but the overall message is to keep it humid!

When drying the slides, the temperature can vary from 20°C to 75°C depending on the protocol. [6,7,13,18,19] A higher temperature can cause the fixative to dry too quickly. A temperature that is too cold could cause an increase in moisture on your slides, as cold air can’t hold as much water as warm air. This could cause your fixative to dry too slowly. As drying time influences chromosome spreading, it is important to find the right combination of temperature and humidity to optimize chromosome spreading for your cells. [17]

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Final Thoughts on Preparing Metaphase Spreads

Once your slides are dry and the chromosomes are fixed in place, they are ready for any downstream applications such as immunostaining. It might be helpful to check your first slide before preparing the next one. That way, you can make any adjustments to your methods (such as distance, humidity, and temperature) based on the chromosome spreading you see under the microscope.

Above all, keep in mind that perfecting your metaphase spreads is an art! Following a protocol step-by-step is a great place to start, but you’ll need to optimize your methods for your needs. If you need a visual protocol or some troubleshooting tips, check out Chromosome Preparation from Cultured Cells by Howe et al. [5]

Do you have any tips for troubleshooting your chromosome harvests? Let us know in the comments.

References:

  1. Wang, N., “Methodologies in cancer cytogenetics and molecular cytogenetics”, American Journal of Medical Genetics vol. 115(3), p118–124, 2002.
  2. Hochstenbach, R., et al., “A survey of undetected, clinically relevant chromosome abnormalities when replacing postnatal karyotyping by Whole Genome Sequencing”, European Journal of Medical Genetics vol.62(9), 103543, 2019.
  3. Bolhaqueiro, A., et al., “Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids”, Nature Genetics vol.51, 824–834, 2019.
  4. Miura, M., et al., “Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation” Stem Cells vol.24(4), 1095-1103, 2006.
  5. Zulkipli, Ihsan N., et al., “Medicinal plants: A potential source of compounds for targeting cell division.” Drug target insights vol.9, 9-19, 2015
  6. Moralli, D., et al., “An improved technique for chromosomal analysis of human ES and iPS cells” Stem cell reviews and reports vol. 7, (2), 471-477, 2011
  7. Howe, B., et al., “Chromosome preparation from cultured cells” Journal of Visualized Experiments vol. 83, 50203, 2014.
  8. Schuck, P.L., et al., “FISHing for damage on metaphase chromosomes”. Balakrishnan L., Stewart J. (eds) DNA Repair. Methods in Molecular Biology, vol. 1999, 2019.
  9. Finkelstein, Yaron et al., “Colchicine poisoning: the dark side of an ancient drug.” Clinical toxicology vol. 48,5, 407-414, 2010.
  10. Rieder, C., et al., “Colcemid and the mitotic cycle” Journal of Cell Science, vol. 102, 387-397, 1992.
  11. Mhaidat, N.M., et al. “Assessment of genotoxicity of vincristine, vinblastine and vinorelbine in human cultured lymphocytes: a comparative study” BJMG vol. 19(1), 13-20, 2016. doi: 10.1515/bjmg-2016-002
  12. Williams, J., Carpentieri, U.” Metaphase Arresting Compounds in Embryos”. Nature vol. 216, 613–614, 1967.
  13. Michelland, S., et al., “A reliable protocol for high-quality metaphase spreads for in situ hybridization”. Technical Tips Online vol. 3, 126-127, 1998.
  14. Kotsarenko, K., et al., “Karyotype changes in long-term cultured tick cell lines”. Sci Rep vol.10, 13443. 2020.
  15. Utsumi, K., et al., “Studies on the Structure of Chromosomes I. The Uncoiling of Chromosomes Revealed by Treatment with Hypotonic Solution: Cell Structure and Function vol. 1, 93-99, 1975.
  16. Bakker, Bjorn et al. “How to count chromosomes in a cell: An overview of current and novel technologies.” BioEssays: news and reviews in molecular, cellular and developmental biology vol. 37,5, 570-577, 2015
  17. Deng, W., et al., “A new method for improving metaphase chromosome spreading” Cytometry, vol. 51A (1), 46-51, 2003.
  18. Purbeck, J.L., et al., “Dynamics of chromosome spreading”. Am J Med Genet; vol. 61(4), 387–393, 1996. doi: 10.1002/(SICI)1096-8628(19960202)61:4<387::AID-AJMG15>3.0.CO;2-O. PMID: 8834053.
  19. Henegariu, O., et al., “Improvements in cytogenetic slide preparation: controlled chromosome spreading, chemical aging and gradual denaturing” Cytometry. vol. 43(2),101-109, 2001. PMID: 11169574.

FAQs

How do you prepare metaphase spread? ›

Preparation of Metaphase Spreads

1. Thaw or passage ES cells onto one well of gelatinized 6 well dish or 35 mm dish approximately 2 to 3 days prior to preparation of chromosome spreads. 2. Feed cells with 3 ml of ES cell media 30 to 60 minutes prior to adding Colcemid.

What is a metaphase spread? ›

Metaphase is a stage during the process of cell division (mitosis or meiosis). Normally, individual chromosomes are spread out in the cell nucleus. During metaphase, the nucleus dissolves and the cell's chromosomes condense and move together, aligning in the center of the dividing cell.

What would be an appropriate reason for preparing a chromosomal spread? ›

The preparation of chromosome spreads is a critical step for a successful analysis of the genetic status of embryonic stem cells by routinely techniques as G-banding, and more sophisticated techniques such as FISH, SKY and CGH.

What are the three main methods of chromosome preparation? ›

There are three main methods of plant chromosome preparation: squashing [9–11], spreading [12, 13] and dropping [14–22]. A squashing method has been the common procedure for chromosome counting in plant cytogenetics during decades.

How do you obtain high quality metaphase spreads for molecular cytogenetics? ›

Place suspension with interphases and metaphases on a slide using a 20-µl micropipette and let air dry for at least 1 hr. Let slide(s) dry overnight at room temperature for best results. The amount of suspension to be placed on the slide depends on the density of the suspension. It may vary between 5 and 15 µl.

How are the chromosomes prepared for analysis? ›

Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible.

What 3 things happen in metaphase? ›

In metaphase, the mitotic spindle is fully developed, centrosomes are at opposite poles of the cell, and chromosomes are lined up at the metaphase plate.

What is the importance of metaphase? ›

There is an important checkpoint in the middle of mitosis, called the metaphase checkpoint, during which the cell ensures that it is ready to divide.

What is a metaphase plate? ›

noun Cell Biology. a plane in the equatorial region of the spindle in dividing cells, along which the chromosomes become arranged during the metaphase.

What is chromosome preparation? ›

Chromosome preparations for cytogenetic analysis are made from dividing cells, either directly from tissue samples (e.g., bone marrow, testis, chorionic villi, neoplastic tissue) or after cell culture (biopsy of skin or almost any other living tissue including amniotic fluid cells).

Why are metaphase cells used for karyotyping? ›

For karyotyping the chromosomes must be isolated from cells in the metaphase, which is the stage of the cell cycle in which the chromosomes assume their characteristic condensed, discrete shape.

What are the steps in karyotyping? ›

Let's take a look at these steps so you can understand what is happening during the time you are waiting for the test.
  1. Sample Collection. ...
  2. Transport to the Laboratory. ...
  3. Separating the Cells. ...
  4. Growing Cells. ...
  5. Synchronizing Cells. ...
  6. Releasing the Chromosomes From Their Cells. ...
  7. Staining the Chromosomes. ...
  8. Analysis.
20 Feb 2022

What is the purpose of pre treatment in chromosome preparation? ›

The pretreatment stage serves to stop the formation of spindles during cell division to keep cells in the metaphase phase and condense the chromosomes so the chromosomes will shorten [9]. Pretreatment solution commonly used is the cold water (0-2 °C), colchicine, 8-hydroxyquinoline, and paradichlorobenzene (PDB) [9].

How are cells prepared for a karyotype? ›

The arrested cells are transferred to a centrifuge tube centrifugation concentrates the cells at the

How is a chromosome spread different from a karyotype? ›

To make a chromosome spread, one blocks the progression of mitosis at metaphase where chromosomes are condensed into the structures we are familiar with. A karyotype analysis is an arrangement of the chromosome spread into the homologous pairs of chromosomes.

How do you become a Cytogeneticist? ›

Becoming a cytogenetic technologist
  1. Obtain a four-year bachelor's degree in cytogenetic technology, biotechnology, biology, or a related science.
  2. While not always required, most employers require certification by the American Society of Clinical Pathologists (ASCP)

How do you make colchicine solution? ›

Prepare a 0.5 % stock solution by dissolving 0.25 g colchicine (Sigma C-9754) in 50 mL distilled water. Colchicine is an alkaloid that is isolated from the plant Colchicum autumnale. Store the solution in a dark bottle at room temperature.

How does fluorescence in situ hybridization work? ›

Fluorescence in situ hybridization (FISH) is a cytogenetic technique developed in the early 1980s. FISH uses fluorescent DNA probes to target specific chromosomal locations within the nucleus, resulting in colored signals that can be detected using a fluorescent microscope.

How do chromosomes spread? ›

Chromosome spreads are usually done in conjunction with immunofluorescence to visualize synaptonemal complex structures or distinct foci. The most common problems encountered in this technique are that the cells just don't burst or that the chromosomes don't spread enough to distinguish individual foci.

What is a chromosome analysis test? ›

What is chromosome analysis? Chromosome analysis is a test to look at the chromosomes in a sample of cells. It can help identify genetic abnormalities as the cause of a condition or disease. The test can count the number of chromosomes present, and look for any structural abnormalities in the chromosomes.

What is the role of fixative in karyotyping? ›

The purpose of fixation is to kill the tissue instantaneously (lethality) without pycnosis, i.e., not causing any distortion of the components to be studied.

What is the characteristics of metaphase? ›

Metaphase is characterised by the alignment of the duplicated chromosomes at the equatorial plate known as the metaphase plate. One of the sister chromatids is attached to one pole of the cell by microtubules attached to its kinetochore and the other sister chromatid gets attached to the opposite pole.

What is the example of metaphase? ›

The traditional illustration of chromosomes is made up of chromosomes in the metaphase phase (karyotype). For classical cytogenetic analyses, the growth of cells in short-term culture before they are arrested in the metaphase cell state with a mitotic inhibitor.

What happens if the metaphase? ›

So in summary metaphase is when the chromosomes of the cell align in the middle of the cell. And

Which structure divides at end of metaphase? ›

DNA at centromere is late duplicating, hence sister chromatids remains attached at this point till the end of metaphase.

What are the stages of metaphase 1? ›

Metaphase I: Homologue pairs line up at the metaphase plate. Anaphase I: Homologues separate to opposite ends of the cell. Sister chromatids stay together. Telophase I: Newly forming cells are haploid, n = 2.

How many chromosomes are in metaphase? ›

Metaphase: During metaphase, each of the 46 chromosomes line up along the center of the cell at the metaphase plate.

Why is it called metaphase? ›

Metaphase com from Greek roots meaning "adjacent or between" and "stage." During this stage, chromosomes stop moving back and forth and are held in the middle of the cell by tube-shaped spirals of protein called microtubules.

What is the metaphase plate made of? ›

To sum it up, the metaphase plate is simply an imaginary plane that exists only during metaphase. It draws a line across the cell that is equidistant from both poles of the cell. This allows the chromosomes a place to be lined up on before they are pulled apart.

What does M phase consist of? ›

Cell division occurs during M phase, which consists of nuclear division (mitosis) followed by cytoplasmic division (cytokinesis). The DNA is replicated in the preceding S phase; the two copies of each replicated chromosome (called sister chromatids) remain glued together by cohesins.

How metaphase plate is formed? ›

Formation of a Metaphase Plate

The formation of the metaphase plate, also the equatorial plate, is dependent on the mechanical forces of the microtubule spindle fibers generated from the centrosome. Chromosomes are evenly aligned at the metaphase plate.

How do chromosomes spread? ›

Chromosome spreads are usually done in conjunction with immunofluorescence to visualize synaptonemal complex structures or distinct foci. The most common problems encountered in this technique are that the cells just don't burst or that the chromosomes don't spread enough to distinguish individual foci.

How many centromeres are in metaphase? ›

In the Metaphase stage of mitosis, each chromosome has 2 chromatids, 1 centromere, 2 kinetochores and 1 unit of DNA.

What happens during metaphase I of meiosis? ›

In metaphase I, the homologous pairs of chromosomes align on either side of the equatorial plate. Then, in anaphase I, the spindle fibers contract and pull the homologous pairs, each with two chromatids, away from each other and toward each pole of the cell. During telophase I, the chromosomes are enclosed in nuclei.

What is a metaphase plate in mitosis? ›

Metaphase plate is an imaginary line which is equidistant from the two poles. During metaphase the chromosomes assemble at the metaphase plate, an imaginary line where the chromosomes are aligned before segregation during Anaphase.

What is a metaphase plate? ›

noun Cell Biology. a plane in the equatorial region of the spindle in dividing cells, along which the chromosomes become arranged during the metaphase.

What happens at the metaphase plate during mitosis? ›

Chromosomes line up at the metaphase plate, under tension from the mitotic spindle. The two sister chromatids of each chromosome are captured by microtubules from opposite spindle poles. In metaphase, the spindle has captured all the chromosomes and lined them up at the middle of the cell, ready to divide.

How is a chromosome spread different from a karyotype? ›

To make a chromosome spread, one blocks the progression of mitosis at metaphase where chromosomes are condensed into the structures we are familiar with. A karyotype analysis is an arrangement of the chromosome spread into the homologous pairs of chromosomes.

What is the difference between mitosis and meiosis? ›

Mitosis is a process where a single cell divides into two identical daughter cells (cell division). facts What is meiosis? Meiosis is a process where a single cell divides twice to produce four cells containing half the original amount of genetic information.

What are 3 differences between mitosis and meiosis? ›

Mitosis consists of one stage whereas meiosis consists of two stages. Mitosis produces diploid cells (46 chromosomes) whereas meiosis produces haploid cells (23 chromosomes). Mitosis produces two identical daughter cells whereas meiosis produces four genetically different daughter cells.

How many chromosomes are in metaphase? ›

Metaphase: During metaphase, each of the 46 chromosomes line up along the center of the cell at the metaphase plate.

How many chromatids are present in metaphase? ›

During metaphase, there are 46 chromosomes composed of two sister chromatids each that align at the metaphase plate. Then, during anaphase, these chromatids are separated and pulled to opposite poles of the cell. This separation results in 92 separate chromatids in the cell, which are considered 92 chromosomes.

Which structure divides at end of metaphase? ›

DNA at centromere is late duplicating, hence sister chromatids remains attached at this point till the end of metaphase.

What 3 things happen in metaphase? ›

In metaphase, the mitotic spindle is fully developed, centrosomes are at opposite poles of the cell, and chromosomes are lined up at the metaphase plate.

What happens in metaphase 2 of meiosis? ›

Metaphase II: The paired chromosomes line up. Anaphase II: The chromatids split at the centromere and migrate along the spindle fibers to opposite poles. Telophase II: The cells pinch in the center and divide again. The final outcome is four cells, each with half of the genetic material found in the original.

What are the stages of metaphase 1? ›

Metaphase I: Homologue pairs line up at the metaphase plate. Anaphase I: Homologues separate to opposite ends of the cell. Sister chromatids stay together. Telophase I: Newly forming cells are haploid, n = 2.

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