Name: Chromosome Preparation From Cultured Cells (Video) | JoVE Journal
Uploaded: 2014-01-28T13:00:00.000+00:00
Duration: 7 min 42 s
Description: Learn chromosome preparation from cultured cells for cytogenetic analysis using lymphocytes and fibroblasts. This method enables G-banding and FISH applications in genetic research.

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5 minutes

Work described in this manuscript was made possible by funding from the Patrick F. Taylor Foundation.

1. Chromosome Harvesting of Adherent Cells
<ol>
	<li>Standard Protocol
	<ol>
		<li>Grow cells according to specific cell culturing conditions. When the cells have reached logarithmic phase (80% confluency), add 10 &#956;l/ml of Colcemid to the cell culture flask. A minimum of 2 x 106 cells is recommended.</li>
		<li>Incubate cells at 37 &#176;C in a 5% CO2 incubator for 45 min. Using a sterile pipette, transfer media from cells into a 15 ml conical tube. Set aside.</li>
		<li>Gently wash the cells by adding 2 ml of HBSS Buffer into the flask. Swirl buffer and then remove using a pipette. Discard.</li>
		<li>Add 1 ml of trypsin, ensuring that it covers the entire surface of the flask. Only leave the cells in trypsin for about 2 min. Once the majority of the cells have detached, pipette the media in the conical tube back onto the cells.</li>
		<li>Transfer the cell suspension in 10 ml aliquots into 15 ml conical tubes. Centrifuge at 200 x g for 10 min. Remove supernatant and resuspend the pellet.</li>
		<li>Add 10 ml of 0.075 M KCl which has been prewarmed to 37 &#176;C to the remaining pellet in the conical tube. Vortex tube at medium speed to mix KCl and cells.</li>
		<li>Incubate cells at 37 &#176;C for 10 min. Centrifuge at 200 x g for 5 min at 25 &#176;C. Remove supernatant (until about 0.5 ml remains) and resuspend pellet.</li>
		<li>Carefully add 5 ml of fresh Carnoy's Fixative (3:1 ratio of methanol:glacial acetic acid) to the cells while vortexing. Then add 5 ml more of fixative without vortexing for a total of 10 ml.</li>
		<li>Centrifuge at 200 x g for 5 min. Remove supernatant and resuspend cells. Add 5 ml of fixative to each tube.</li>
		<li>Centrifuge at 200 x g for 5 min. Remove supernatant and resuspend cells. Add 5 ml of fixative to each tube. The cells can now be stored in 4 &#176;C for up to one year.</li>
	</ol>
	</li>
	<li>Modification to Protocol: Chromosome harvesting of lymphoblastoid cell lines
	<ol>
		<li>Grow cells according to specific cell culturing conditions. (Use flask appropriate for the amount of cells being cultured). When the cells have reached logarithmic phase (approximately 2 days after cells are split), add 10 &#956;l/ml of Colcemid to the cell culture flask.</li>
	</ol>
	</li>
	<li>Modification to Protocol: Chromosome harvesting from whole blood 
	Phytohemagglutinin (PHA), a lectin derived from the red kidney bean, is a powerful mitogen for human T-cells16. 72 hr after the addition of PHA to the culture, about 45% of cells are in S phase. This represents the peak mitotic activity, and is the optimum point at which to harvest for chromosome studies. Other mitogens such as pokeweed (1-10 &#956;g/ml) may be used when analyzing B cells17,18.
	<ol>
		<li>Collect at least 1 ml of whole blood in a green top sodium heparin tube. Use within 3 days after collection. Store blood at RT until ready to use.</li>
		<li>Aliquot 0.25 ml of whole blood in 10 ml of complete RPMI media containing L-glutamine (20% fetal bovine serum, 1% Penicillin/streptomycin, 1% fungizone, and 1% PHA). Culture at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>Modification to Protocol: Chromosome harvesting from bone marrow Cytogenetic analysis on bone marrow is helpful in many malignant hematologic disorders as the observation of a chromosomally abnormal clone may be diagnostic for a specific type of leukemia. Chromosome studies are also used to assess disease progression such as the onset of blast crisis and response to treatment. Since bone marrow cells are actively dividing, no mitogen stimulation is necessary9,19. For acute leukemias 24 and 48 hr cultures are also set up.
	<ol>
		<li>Collect at least 1 ml of bone marrow in a green top sodium heparin tube. Aliquot 0.25 ml of bone marrow in 10 ml of complete RPMI media containing L-glutamine (20% fetal bovine serum, 1% Penicillin/streptomycin, 1% fungizone). Culture at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
</ol>
2. Slide Preparation and Solid Staining
A rapid evaluation of one representative slide will provide information on the quality of the harvest before continuing to further procedures such as G-banding, FISH, CGH, or SKY. Some laboratories prefer to solid stain the cells for rapid harvesting and chromosome assessment. Alternatively, a phase contrast microscope may be used for this analysis. Slides are best prepared when the humidity is approximately 50% and the temperature ambient (20-25 &#176;C).
<ol>
	<li>Centrifuge the cells at 200 x g for 5 min at 25 &#176;C. Remove the supernatant until only 0.3-0.5 ml remains.</li>
	<li>After gently resuspending the pellet, pipette three drops of the cell suspension from a distance of about 2 in onto a slide which is tilted at an angle of about 45&#176; and allow the suspension to roll across the slide. Add one large drop of fresh Carnoy's Fixative to the slide.</li>
	<li>Dry the back of the slide on a paper towel and then sit the slide out to dry for at least 10 min. The slide should be completely dry.</li>
	<li>Prepare fresh Giemsa Staining Solution (3:1 ratio of Gurr Buffer and Giemsa Stain). Place the slides on a staining rack. Cover the entire slide in the Giemsa staining solution. Let the slides remain in the staining solution for 5 min. Rinse slides with distilled water, drain, and allow to air dry.</li>
	<li>Add 4 drops of Permount and a cover slip to the slide. Make sure there are no bubbles under the coverslip. The excess Permount can be removed with a paper towel.</li>
	<li>Analyze cells with a light microscope under 10X and 100X magnification. If the metaphase cells are abundant and well spread, the remaining slides can be used for other experimental procedures.</li>
</ol>
3. G-Banding Using Trypsin and Giemsa (GTG)
Trypsin is a proteolytic enzyme which denatures euchromatic histones in DNA regions with higher transcriptional activity resulting. Following Giemsa staining, these regions will appear as light bands. Highly condensed chromatin with little or no transcriptional activity (heterochromatin) will have a large portion of its histones protected from the trypsin and will therefore stain darkly following Giemsa staining. It is essential to initially G-band one slide to monitor the conditions and adjust the trypsin timing if necessary for the subsequent slides.
<ol>
	<li>Add the following solutions to 4 Coplin jars. Jar #1= 30 ml of 1x HBSS and 4 ml of 10x trypsin (0.5%) 
	Jar #2= 50 ml of 1x HBSS 
	Jar #3= 45 ml of 1x HBSS and 5 ml of fetal bovine serum 
	Jar #4= 50 ml of 1x HBSS</li>
	<li>Immerse each slide in Jar #1 for 5 sec, quick rinse in Jar #2, leave each slide in Jar #3 for at least 30 sec, quick rinse in Jar #4 then allow slides to dry.</li>
	<li>Prepare fresh Giemsa Staining Solution (3:1 ratio of Gurr Buffer and Giemsa Stain). Place the slides on a staining rack. Cover the entire slide in the Giemsa staining solution. Let the slides remain in the staining solution for 5 min.</li>
	<li>Rinse slides in distilled water in the same order that they were stained. Allow the slides to dry for about 10 min. The slide should be completely dry.</li>
	<li>Add 4 drops of Permount and a cover slip to the slide. Make sure there are no bubbles under the coverslip. The excess Permount can be removed with a paper towel.</li>
	<li>Analyze cells with a light microscope under 10X and 100X magnification. Adjust trypsin timing of the rest of the slides based on the results of the first slide.</li>
</ol>

<ol><li>Driscoll, D., Gross, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prenatal+screening+for+aneuploidy%5BTitle%5D)+AND+%22New+Engl.+J.+Med%22%5BJournal%5D)">Prenatal screening for aneuploidy.</a> New Engl. J. Med. 360, 2556-2562 (2009).</li>
<li>Nussbuam, R., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNussbuam%2C+R+%22Genetics+in+Medicine%3A+7th+Edition%22">Genetics in Medicine: 7th Edition</a>. , Saunders/Elsevier. Philadelphia. 76 (2007).</li>
<li>Ljunger, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosomal+abnormalities+in+first-trimester+miscarriages%5BTitle%5D)+AND+%22Acta+Obstet.+Gynecol.+Scand%22%5BJournal%5D)">Chromosomal abnormalities in first-trimester miscarriages.</a> Acta Obstet. Gynecol. Scand. 84 (11), 1103-1107 (2005).</li>
<li>Kwinecka-Dmitri, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Frequency+of+chromosomal+aberrations+in+material+from+abortions%5BTitle%5D)+AND+%22Ginecol.+Pol%22%5BJournal%5D)">Frequency of chromosomal aberrations in material from abortions.</a> Ginecol. Pol. 81 (12), 896-901 (2010).</li>
<li>Reddy, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stillbirth+classification%E2%80%94Developing+an+international+consensus+for+research%3A+Executive+summary+of+a+national+institute+of+child+health+and+human+development+workshop%5BTitle%5D)+AND+%22Obstet.+Gynecol%22%5BJournal%5D)">Stillbirth classification—Developing an international consensus for research: Executive summary of a national institute of child health and human development workshop.</a> Obstet. Gynecol. 114 (4), 901-914 (2009).</li>
<li>Pierpoint, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+basis+for+congenital+heart+defects%3A+Current+knowledge%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Genetic basis for congenital heart defects: Current knowledge.</a> Circulation. 115 (23), 3015 (2007).</li>
<li>Kaneshiro, N., Zieve, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Down+Syndrome%3A+Trisomy+21.%5BTitle%5D)+AND+%22PubMed+Health%22%5BJournal%5D)">Down Syndrome: Trisomy 21.</a> PubMed Health. , (2010).</li>
<li>Cin, P. D. Current Protocols in Human Genetics. , (2003).</li>
<li>Wang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methodologies+in+cancer+cytogenetics+and+molecular+cytogenetics%5BTitle%5D)+AND+%22Am.+J.+Med.+Genet%22%5BJournal%5D)">Methodologies in cancer cytogenetics and molecular cytogenetics.</a> Am. J. Med. Genet. 115 (3), 118-124 (2002).</li>
<li>Miura, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accumulated+Chromosomal+Instability+in+Murine+Bone+Marrow+Mesenchymal+Stem+Cells+Leads+to+Malignant+Transformation%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Accumulated Chromosomal Instability in Murine Bone Marrow Mesenchymal Stem Cells Leads to Malignant Transformation.</a> Stem Cells. 24 (4), 1095-1103 (2006).</li>
<li>Izadpanah, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+In+vitro+Expansion+Alters+the+Biology+of+Adult+Mesenchymal+Stem+Cells%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Long-term In vitro Expansion Alters the Biology of Adult Mesenchymal Stem Cells.</a> Cancer Res. 68 (11), 4229-4238 (2008).</li>
<li>Dekel-Naftali, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Screening+of+human+pluripotent+stem+cells+using+CGH+and+FISH+reveals+low-grade+mosaic+aneuploidy+and+a+recurrent+amplification+of+chromosome+1q%5BTitle%5D)+AND+%22Eur.+J.+Hum.+Genet%22%5BJournal%5D)">Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q.</a> Eur. J. Hum. Genet. 20 (12), 1248-1255 (2012).</li>
<li>Moralli, D., Yusuf, M., Mandegar, M., Khoja, S., Monaco, Z. L., Volpi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+Improved+Technique+for+Chromosomal+Analysis+of+Human+ES+and+iPS+Cells%5BTitle%5D)+AND+%22Stem+Cell+Rev.+Rep%22%5BJournal%5D)">An Improved Technique for Chromosomal Analysis of Human ES and iPS Cells.</a> Stem Cell Rev. Rep. 7 (2), 471-477 (2011).</li>
<li>Bayani, J., Squire, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Traditional+banding+of+chromosomes+for+cytogenetic+analysis%5BTitle%5D)+AND+%22Curr.+Protoc.+Cell+Biol%22%5BJournal%5D)">Traditional banding of chromosomes for cytogenetic analysis.</a> Curr. Protoc. Cell Biol. 22, (2004).</li>
<li>Speicher, M. R., Carter, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+new+cytogenetics%3A+blurring+the+boundaries+with+molecular+biology%5BTitle%5D)+AND+%22Nat.+Rev.+Genet%22%5BJournal%5D)">The new cytogenetics: blurring the boundaries with molecular biology.</a> Nat. Rev. Genet. 6 (10), 782-792 (2005).</li>
<li>Benn, P., Delach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+Lymphocyte+Culture+and+Chromosome+Analysis%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">Human Lymphocyte Culture and Chromosome Analysis.</a> Cold Spring Harb Protoc. , (2010).</li>
<li>Muul, L. M., Heine, G., Silvin, C., James, S. P., Candotti, F., Radbruch, A., Worm, M. Measurement of Proliferative Responses of Cultured Lymphocytes, Curr. Protoc. Immunol. , (2011).</li>
<li>Barch, M. J., Knutsen, T., Spurbeck, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBarch%2C+MJ+%22The+Association+of+Genetic+Technologists+Cytogenetics+Laboratory+Manual.+San+Francisco%3A+3rd+edition%22">The Association of Genetic Technologists Cytogenetics Laboratory Manual. San Francisco: 3rd edition</a>. , ACT. Houston, TX. (1997).</li>
<li>Dewald, G. W., Ketterling, R. P., Wyatt, W. A., Stupca, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytogenetic+studies+in+neoplastic+hematologic+disorders%5BTitle%5D)+AND+%22In+Clinical+Laboratory+Medicine%2C+2nd+edition%22%5BJournal%5D)">Cytogenetic studies in neoplastic hematologic disorders.</a> In Clinical Laboratory Medicine, 2nd edition. McClatchey, K. D. , Williams & Wilkens. Baltimore. 658-685 (2002).</li>
<li>Kibe, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IL-7R%CE%B1+deficiency+in+p53%28null%29+mice+exacerbates+thymocyte+telomere+erosion+and+lymphomagenesis%5BTitle%5D)+AND+%22Cell+Death+Different%22%5BJournal%5D)">IL-7Rα deficiency in p53(null) mice exacerbates thymocyte telomere erosion and lymphomagenesis.</a> Cell Death Different. 19 (7), 1139-1151 (2012).</li>
<li>Tsien, F., Morava, E., Talarski, A., Marble, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phenotypic+features+of+a+boy+with+trisomy+of+16q22--%3Eqter+due+to+paternal+Y%3B+16+translocation%5BTitle%5D)+AND+%22Clin.+Dysmorphol%22%5BJournal%5D)">Phenotypic features of a boy with trisomy of 16q22-->qter due to paternal Y; 16 translocation.</a> Clin. Dysmorphol. 4 (4), 177-181 (2005).</li>
<li>Tsien, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prolonged+culture+of+normal+chorionic+villus+cells+yields+ICF+syndrome-like+chromatin+decondensation+and+rearrangements%5BTitle%5D)+AND+%22Cytogenet.+Genome+Res%22%5BJournal%5D)">Prolonged culture of normal chorionic villus cells yields ICF syndrome-like chromatin decondensation and rearrangements.</a> Cytogenet. Genome Res. 8 (1), 13-21 (2002).</li>
<li>McGrattan, P., Campbell, S., Cuthbert, R., Jones, F., McMullin, M., Humphreys, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integration+of+conventional+cytogenetics+%28G-banding%29%2C+comparative+genomic+hybridization+%28CGH%29+and+interphase+fluorescence+in+situ+hybridization+%28i-FISH%29+for+the+detection+of+genomic+rearrangements+in+acute+leukemia%5BTitle%5D)+AND+%22J.+Clin.+Pathol%22%5BJournal%5D)">Integration of conventional cytogenetics (G-banding), comparative genomic hybridization (CGH) and interphase fluorescence in situ hybridization (i-FISH) for the detection of genomic rearrangements in acute leukemia.</a> J. Clin. Pathol. 61 (8), 903-908 (2008).</li>
</ol>

The authors have nothing to disclose.

We have utilized the present procedure for chromosome isolation from cells of various organisms, including various cell types obtained from human, Rhesus macaques, rats, and mice11,20-22. The standard protocol is provided, but certain key steps and variables may need to be adjusted for these diverse types of cells. Several specific steps are crucial in both the chromosomal preparation and G-banding to ensure the best possible quality of the results. One of the variables which can affect the assay is the Colcem...

Chromosome analysis is a conventional technique utilized worldwide to diagnose chromosome instability and rearrangements leading to genetic disorders and malignancy1,2,8,9. In addition, a higher resolution for the diagnosis and research of constitutional and cancer-acquired genetic abnormalities can be achieved with the combination of the classical cytogenetic procedures and molecular cytogenetic methodologies such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15. More recently, these techniques have been utilized for the evaluation of chromosome instability associated with stem cell research. Karyotypic abnormalities such as aneuploidy of long-term cultured embryonic cells (ES) and adult stem cells of various organisms have been reported by multiple laboratories. Recent evidence supports that some cell lines are inherently more inclined to chromosome instability regardless of culture conditions. For this reason, when establishing and/or maintaining human, mouse, or Rhesus stem cell lines, chromosome analysis is recommended as part of the quality control process. Many reports describe the increasing interest in the use of routine and molecular cytogenetics to monitor the chromosomal stability of stem cells and malignant cells or various organisms in culture10-13. These protocols are increasingly being used by nongenetic laboratories for rapid chromosomal assessment of their cultured cells13. We present our basic procedures for chromosome preparation from various cell types, which can be applied for both clinical and research purposes and cells derived from various organisms.

<table><tbody><tr><td>KaryoMAX Colcemid</td><td>Gibco</td><td>15212-012</td><td></td></tr><tr><td>HBSS Buffer</td><td>Gibco</td><td>24020-117</td><td></td></tr><tr><td>0.5% Trypsin EDTA 10x</td><td>Gibco</td><td>15400-054</td><td></td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-100</td><td></td></tr><tr><td>Buffer Tablets &quot;GURR&quot;</td><td>Gibco</td><td>10580-013</td><td></td></tr><tr><td>Geimsa Stain</td><td>Ricca Chemical Company</td><td>3250-16</td><td></td></tr><tr><td>Centrifuge 5810</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Methanol</td><td>Caledon Laboratory Chemicals</td><td>6700130</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Krackeler Scientific Inc.</td><td>11-9508-05</td><td></td></tr></tbody></table>

chromosome preparation from cultured cells

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
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. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

High quality metaphase spreads are essential for chromosome analysis. A successful assay yields chromosomes which are well spread and of suitable chromosome morphology. Properly G-banded chromosomes contain the characteristic light and dark banding patterns.
<img alt="Chromosome spread diagram, karyotype analysis, genetic study visualization." fo:content-width="5in" fo:src="/files/ftp_upload/50203/50203fig1highres.jpg" src="/files/ftp_upload/50203/50203fig...

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

The overall goal of this procedure is to harvest and prepare chromosomes for GB banding and molecular cytogenetic tests. First, culture the cells to logarithmic phase. Then harvest the chromosomes with smid, a hypotonic and a fixative.Drop the cell suspension into slides. Stain one slide. As a monitor of the chromosomal preparation, proceed to GB banding or molecular techniques of the remaining slides.As a final step in GB banding, analyze the chromosomes by light microscopy and karyotyping. Ultimately, the chromosomes and nuclei will be available for karyotyping or fish ish. I first had the idea for publishing this method because of the high demand from other researchers requesting assistance in the detection of chromosome instability in different types of cells from various organisms and for subsequent molecular cytogenetic testing.This technique is essential in the diagnosis and ultimately prognosis of many congenital genetic disorders including cancer, because it allows for the visualization of chromosome rearrangements, which are diagnostic. For these disorders, Though this method can provide insight into the chromosome rearrangements found in various types of human cells. It can also be applied to cells from other organisms such as Resus Maca rats and mice Culture.2 million adherent cells to 80%con fluency and add 10 microliters of 10 micrograms per milliliter. Col per milliliter of cells. Incubate the cells at 37 degrees Celsius in a 5%CO2 incubator for 45 minutes.Then using a sterile pipette, transfer the media from the culture into a 15 milliliter conical tube and reserve for later To wash the cells, add two milliliters of HBSS into the flask, swirl and remove the buffer. Next, add one milliliter of trypsin, ensuring it covers the entire surface of the flask. Once the majority of the cells have detached, pipette the media in the conical tube back onto the cells.Aliquot 10 milliliters of the cell suspension into 15 milliliter conical tubes. Centrifuge at 200 Gs for 10 minutes. Remove the supernat.Re suspend the pellet in 10 milliliters of 75 millimolars potassium chloride, which will be prewarm to 37 degrees Celsius after a 10 minute incubation of 37 degrees Celsius. Centrifuge at 200 Gs for five minutes. At 25 degrees Celsius, remove all but 0.5 milliliters of the supernatant and resuspend the pellet Carefully.Add five milliliters of fresh car noise fixative to the cells. Then add five milliliters, more of the fixative and vortex centrifuge at 200 Gs for five minutes. Resus, suspend each cell pellet in five milliliters of fixative.Store the cells at four degrees for up to one year. Centrifuge the cells at 200 Gs for five minutes. At 25 degrees Celsius.Remove the sup natant until only 0.3 to 0.5 milliliters remains after Gently resus suspending the pellet. Pipette three drops of the cell suspension from a distance of about two inches onto a slide which is tilted at an angle of about 45 degrees. And allow the suspension to roll across the slide.Add one large drop of fresh car noise, fixative to the slide. Drive the back of the slide on a paper towel, and then sit the slide out to dry completely. Prepare fresh gim sustaining solution.Place the slides on a staining rack. Cover the entire slide in the gim sustaining solution. After five minutes, rinse the slides with distilled water drain and allow to air dry.Add four drops of perm mount to the slide and place a cover slip ensuring there are no bubbles under the cover slip. Remove the excess per mount with a paper towel. Analyze the cells with a light microscope under 10 x and 100 x magnification.If the metaphase cells are abundant and well spread, the remaining slides can be used for other experimental procedures. Add 50 milliliters of the treatment solutions to four Copeland jars. Immerse each slide in jar number one for five seconds after.Quickly rinsing the slide in jar number two. Leave it in jar number three for at least 30 seconds. Rinse in jar number four, then allow the slides to dry.Prepare fresh GIM sustaining solution. Place the slides on a staining rack. Cover the entire slide in the gim sustaining solution for five minutes.Rinse the slides and distilled water in the same order that they were stained. Then dry for at least 10 minutes. Place a cover slip using perm mount as shown earlier.Then analyze the cells under a light microscope for the remaining slides. Adjust the tripsin incubation time. Based on the results of the first slide.A successful assay yields chromosomes that are well spread and of suitable chromosome morphology.Properly. GB banded chromosomes contain the characteristic light and dark banding patterns. After watching this video, you should have a good understanding of how to harvest chromosomes for GB banding and further molecular cytogenetic testing in the elucidation of chromosome instability.Once mastered, this harvesting technique can be done in about three hours if it is performed properly. For the GB banding procedure, it's important to initially test the tripsin incubation time on one slide before proceeding to GB band. The rest of the slides.

chromosome-preparation-from-cultured-cells

Research

JoVE Journal

Biology

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

Combining DNA halo preparations with fluorescence in situ hybridization enables high-resolution analysis of genomic interactions with the nucleoskeleton. Attached genome leads to hybridized fluorescent signals located within the residual extracted nuclei, whereas non-attached genome is in the halo of DNA surrounding the residual nuclei.

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

Hi-C 3.0 is an improved Hi-C protocol that combines formaldehyde and disuccinimidyl glutarate crosslinkers with a cocktail of DpnII and DdeI restriction enzymes to increase the signal-to-noise ratio and the resolution of chromatin interaction detection.

Capturing Chromosome Conformation Across Length Scales

This protocol describes the Capture Hi-C method used to characterize the 3D organization of megabased-sized targeted genomic regions at high-resolution, including boundaries of topologically associating domains (TADs) and long-range chromatin interactions between regulatory and other DNA sequence elements.

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

Learn chromosome preparation from cultured cells for cytogenetic analysis using lymphocytes and fibroblasts. This method enables G-banding and FISH applications in genetic research.

Chromosome Preparation From Cultured Cells

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