eoe sub articles

EoE Sub Articles

1. Gradient Centrifugation
<ol>
	<li>Add 200 &#181;L of 50% iodixanol solution (Table 1) to give a final concentration of 25% iodixanol. Mix well 10 times with a pipette set on 300 mL.</li>
	<li>Add 300 &#181;L of 29% iodixanol solution (Table 2) under the 25% mixture. Use a P1000 fine tip to avoid mixture of the layers.</li>
	<li>Add 300 &#181;l of 35% iodixanol solution (Table 3) under the 29% mixture. Use a P1000 fine tip to avoid mixture of the layers. 
	CAUTION: This step requires gradual removal of the pipette tip during pipetting to avoid excessive volume displacement.</li>
	<li>Place the samples in a swinging bucket centrifuge, spin for 20 min at 3,500 x&#160;g&#160;at 4&#176;C with the brake off.</li>
	<li>Gently remove the samples without shaking and observe under light. A clear white band of 95% pure nuclei should be visible between the second and third layer (Figure 1).</li>
</ol>
2. Isolation of Nuclei
<ol>
	<li>Aspirate the top layers until the white nuclei band at the interphase of 29%&#8211;35%.</li>
	<li>Collect the nuclei band in a 200 mL volume, transfer to a fresh tube, and filter with a 20 &#181;m filter (Table of Materials). 
	NOTE: The nuclei do not need to be resuspended prior to filtration.</li>
	<li>Check under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane. Nuclei need to be round, and the nuclear membrane should not be distorted.</li>
	<li>Count nuclei using Trypan blue staining on a hemocytometer and aliquot nuclei for snRNA-seq/snATAC-seq.</li>
</ol>
Table 1: Preparation of 6x Homogenization Buffer Stable Master Mix
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">6x Homogenization Buffer Stable Master Mix</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Final Conc.</td>
			<td>Vol for 100 (mL)</td>
		</tr>
		<tr>
			<td>1 M CaCl&#8322;</td>
			<td>30 mM</td>
			<td>3.0</td>
		</tr>
		<tr>
			<td>1 M Mg(Ac)&#8322;</td>
			<td>18 mM</td>
			<td>1.8</td>
		</tr>
		<tr>
			<td>1 M Tris pH 7.8</td>
			<td>60 mM</td>
			<td>6.0</td>
		</tr>
		<tr>
			<td>H&#8322;O</td>
			<td></td>
			<td>89.2</td>
		</tr>
		<tr>
			<td colspan="3">Keep at room temperature, avoid direct exposure to light</td>
		</tr>
	</tbody>
</table>


Table 2: Preparation of 1 M sucrose
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 M Sucrose</td>
		</tr>
		<tr>
			<td>34.23 g of sucrose</td>
		</tr>
		<tr>
			<td>Dissolve in 78.5 mL of water</td>
		</tr>
		<tr>
			<td>Fill up to 100 mL with water</td>
		</tr>
	</tbody>
</table>


Table 3: Preparation of 6x Homogenization Buffer Unstable Solution (650 &#181;L per sample)

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">6x Homogenization Buffer Unstable Solution (650 mL per sample)</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Final Conc.</td>
			<td>Vol per sample (&#181;L)</td>
		</tr>
		<tr>
			<td>6x Homogenization Buffer Stable</td>
			<td>6x</td>
			<td>648.84</td>
		</tr>
		<tr>
			<td>100 mM PMSF (Phenylmethylsulfonyl fluoride)</td>
			<td>0.1 mM</td>
			<td>1.08</td>
		</tr>
		<tr>
			<td>14.3 M &#946;-mercaptoethanol</td>
			<td>1 mM</td>
			<td>0.08</td>
		</tr>
	</tbody>
</table>

<table><tbody><tr><td>EDTA (0.5 M)</td><td>Thermo Scientific</td><td>R1021</td><td></td></tr><tr><td>Falcon 15 mL Conical Centrifuge&lt;br/&gt; Tubes</td><td>Fisher Scientific</td><td>352096</td><td></td></tr><tr><td>Iodixanol (aka Optiprep)</td><td>Stem cell technologies</td><td>07820</td><td></td></tr><tr><td>NP-40</td><td>Abcam</td><td>ab142227</td><td></td></tr><tr><td>Safe lock tubes 1.5 mL</td><td>Eppendorf</td><td>0030120086</td><td></td></tr><tr><td>Safe lock tubes 2.0 mL</td><td>Eppendorf</td><td>0030120094</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>Wide Bore pipette tips (1000 &amp;micro;L)</td><td>Thermo Fisher Scientific</td><td>2079GPK</td><td></td></tr><tr><td>Wide Bore pipette tips (200 &amp;micro;L)</td><td>Thermo Fisher Scientific</td><td>2069GPK</td><td></td></tr></tbody></table>

gradient centrifugation based purification of nuclei: a technique to obtain ultrapure fraction of intact nuclei using density gradient centrifugation

Source: Narayanan, A. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/61542/nuclei-isolation-from-fresh-frozen-brain-tumors-for-single-nucleus">Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq.</a> J. Vis. Exp. (2020)
In this video, we describe a simple and efficient protocol using gradient centrifugation to isolate single nuclei. Once isolated, these nuclei can be used for sequencing and genome analysis.

<img alt="Figure 1" src="/files/ftp_upload/20617/20617_Figure1.jpg" /> 
Figure 1: Flow chart for nuclei isolation.&#160;The flow chart provides a brief outlook on the steps involved in the isolation of single nuclei from a fresh frozen glioma tissue. Representative images for the tumor sample and the nuclear band after Iodixanol/sucrose gradient (circled with red dotted line) are shown.

Gradient Centrifugation Based Purification of Nuclei: A Technique to Obtain Ultrapure Fraction of Intact Nuclei Using Density Gradient Centrifugation

Source: Narayanan, A. et al. Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq. J. Vis. Exp. (2020)
In this video, ...

To isolate and purify intact nuclei, begin by taking a crude nuclei fraction in a tube. The fraction contains intact nuclei along with broken nuclei fragments and debris.
Mix this fraction with a suitable density gradient medium such that it forms a suspension of the lowest concentration.
Now, layer solutions of increasing densities of the same gradient medium beneath the pre-prepared crude nuclei fraction to generate a multi-step discontinuous density gradient. This step allows the crude nuclei fraction to form a band at the top of the gradient.
Next, centrifuge the tube at a low speed.
Based on the differences in shape and mass, intact nuclei migrate differently compared to debris and nuclear fragments. This results in intact nuclei forming a separate band.
After centrifugation, locate the distinct band consisting of intact nuclei at the interface of density gradient solutions.
Using a pipette, aspirate the top layers of the gradient to facilitate access to the pure nuclei band. Collect the nuclei-containing band into a fresh tube.
Visualize these nuclei under a light microscope. Intact nuclei appear perfectly round with an intact nuclear membrane surrounding them.
Finally, store the pure nuclear fraction for further analysis.

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Universidad de Cartagena UDEC

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1.8K Views. Source: Narayanan, A. et al. Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq. J. Vis. Exp. (2020) In this video, we describe a simple and efficient protocol using gradient centrifugation to isolate single nuclei. Once isolated, these nuclei can be used for sequencing and genome analysis. 

Video: Gradient Centrifugation Based Purification of Nuclei: A Technique to Obtain Ultrapure Fraction of Intact Nuclei Using Density Gradient Centrifugation - Concept

Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are available to quantify and compare capsule production between different strains or mutants, there is no widely used method for sorting bacteria based on how much capsule they produce. We have developed a method to separate bacteria by capsule amount, using a discontinuous density gradient. This method is used to compare capsule amounts semi-quantitatively between cultures, to isolate mutants with altered capsule production, and to purify capsulated bacteria from complex samples. This method can also be coupled with transposon-insertion sequencing to identify genes involved in capsule regulation. Here, the method is demonstrated in detail, including how to optimize the gradient conditions for a new bacterial species or strain, and how to construct and run the density gradient.

We demonstrate the use of discontinuous density gradients to separate bacterial populations based on capsule production. This method is used to compare capsule amount between cultures, isolate mutants with a specific capsule phenotype, or to identify capsule regulators. Described here is the optimization and running of the assay.

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are ...

JoVE Journal

Genetics

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent pathways. Until recently, thermogenic adipocytes were considered a homogeneous population. However, recent studies have indicated that there are multiple subtypes or subpopulations that are distinct in developmental origin, substrate use, and transcriptome. Despite advances in single-cell genomics, unbiased decomposition of adipose tissues into cellular subtypes has been challenging because of the fragile nature of lipid-filled adipocytes. The protocol presented was developed to circumvent these obstacles by effective isolation of single nuclei from adipose tissue for downstream applications, including RNA sequencing. Cellular heterogeneity can then be analyzed by RNA sequencing and bioinformatic analyses.

This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Gradient Centrifugation Based Purification of Nuclei: A Technique to Obtain Ultrapure Fraction of Intact Nuclei Using Density Gradient Centrifugation

Transcript

Gradient Centrifugation Based Purification of Nuclei: A Technique to Obtain Ultrapure Fraction of Intact Nuclei Using Density Gradient Centrifugation

Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements