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>

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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 border="0" cellpadding="0" cellspacing="0" width="662">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">KaryoMAX Colcemid</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:145px;">15212-012</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS Buffer</td>
			<td>Gibco</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Trypsin EDTA 10x</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buffer Tablets &#34;GURR&#34;</td>
			<td>Gibco</td>
			<td>10580-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Geimsa Stain</td>
			<td>Ricca Chemical Company</td>
			<td>3250-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5810</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Caledon Laboratory Chemicals</td>
			<td>6700130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glacial Acetic Acid</td>
			<td>Krackeler Scientific Inc.&#160;</td>
			<td>11-9508-05</td>
			<td></td>
		</tr>
	</tbody>
</table>

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="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50203/50203fig1highres.jpg" src="/files/ftp_upload/50203/50203fig1.jpg" /> 
Figure 1.&#160;A successfu...

Watch this Scientific Journal Video about Chromosome Preparation From Cultured Cells at JoVE.com

Chromosome Preparation From Cultured Cells

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 ...

chromosome-preparation-from-cultured-cells

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Biology

80.7K Aufrufe.  Louisiana State University Health Science Center.  Chromosomen von lebenden Zellen, wie Lymphozyten oder Fibroblasten der Haut isoliert werden und aus Organismen, einschließlich Menschen oder Mäusen. Diese Chromosomen-Präparate können weitere für die Routine G-Banding und molekulare zytogenetischen Verfahren wie Fluoreszenz-in-situ-Hybridisierung (FISH), komparative genomische Hybridisierung (CGH), und spektrale Karyotypisierung (SKY) genutzt werden. 

Serie: Chromosome Preparation From Cultured Cells

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

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.

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...