Authors acknowledge the assistance of Ander Lopez and Miren Telletxea for video recording and editing. ACR is funded by an Ikerbasque fellowship and a research project from Asociaci&#243;n de Celiacos Madrid (ACM). JRB is funded by Project ISCIII-PI16/00258 and co-funded by the European Union ERDF/ESF "A way to make Europe". IS is funded by a research project grant 2015111068 of the Basque Department of Health. IRG and AJM are supported by predoctoral fellowship grants from the UPV/EHU and the Basque Department of Education, respectively.

This study was approved by the Cruces University Hospital Ethics Board and analyses were performed after informed consent was obtained from all subjects or their parents.
1. Biopsy Collection
NOTE: Biopsy specimens from the distal duodenum of patients are obtained during routine diagnosis endoscopy managing biopsies that range between 2 and 10 mg in weight. Biopsied tissue is a complex tissue composed of different cell types including epithelial, immune and endothelial cells. Clinical gastroenterologists perform the endoscopy following established clinical guidelines.
<ol>
	<li>Place the biopsy on a sterile filter paper just after endoscopy.</li>
	<li>Use forceps to transfer the biopsies in 1.5 mL tubes, soak them with PBS, and place it into a cryotube.</li>
	<li>Keep tubes on ice until they arrive at the laboratory.
	<ol>
		<li>Start processing of fresh biopsies immediately.</li>
	</ol>
	</li>
	<li>For frozen biopsies, flash freeze the biopsy containing tubes and store in liquid nitrogen until use. 
	NOTE: It is important to keep the sample frozen until the cold supplemented buffer 1 is added.</li>
</ol>
2. Buffer Preparation
Note: Before starting, supplement the buffers as follows.
<ol>
	<li>Add 1 mM DTT (final concentration) and protease and phosphatase inhibitor cocktail (1x final concentration) to the hypotonic buffer 1 and hypertonic buffer 2 (Table 1).
	<ol>
		<li>Prepare 1.5 mL of buffer 1 and 150 &#181;L of buffer 2 per sample. DTT will prevent the oxidation of the samples.</li>
	</ol>
	</li>
	<li>Prepare nuclei wash buffer by adding 1% detergent (nonyl phenoxypolyethoxylethanol) to a final concentration of 0.1% to the already supplemented buffer 1. Prepare 1 mL of nuclei wash buffer per sample.</li>
</ol>
3. Biopsy Homogenization
<ol>
	<li>Fresh Biopsies
	<ol>
		<li>Place the disposable pestle in the motorized mixer. Ensure that the pestle reaches the bottom of the tube.</li>
		<li>Take the biopsy out from the cryotube and place it in a 1.5 mL tube with a pipette tip.</li>
		<li>Add 200 &#181;L of supplemented buffer 1 to the 1.5 mL tube.</li>
		<li>Homogenize the biopsy with the pestle until the tissue is completely disrupted and keep it on ice.</li>
		<li>Once all the biopsies are homogenized proceed with step 4. 
		Note: Disposable pestles must be changed between biopsies.</li>
	</ol>
	</li>
	<li>Frozen Biopsies 
	CAUTION: Handle liquid nitrogen carefully; contact of liquid nitrogen with the skin or eyes may cause serious freezing injury. Protect hands and eyes always when working with liquid nitrogen .
	<ol>
		<li>Take some liquid nitrogen in a box to store the cryotubes retrieved from the nitrogen tank.</li>
		<li>Find the biopsy in the nitrogen tank and place the cryotube in the box with the liquid nitrogen. Repeat this step for all the biopsies.</li>
		<li>Transfer the biopsy from the cryotube to a 1.5 mL tube. 
		NOTE: Biopsies will be attached to the frozen tube. Push the biopsy with a sterile pipette tip so it will detach from the tube.</li>
		<li>Add 200 &#181;L of supplemented buffer 1.</li>
		<li>Homogenize the tissue using the pestle and mortar until the tissue is completely disrupted, maintaining the temperature at 4 &#176;C throughout all the procedure. 
		Note: Do not let biopsies thaw before adding the buffer.</li>
	</ol>
	</li>
</ol>
4. Cytoplasm Isolation
<ol>
	<li>Incubate the homogenized biopsy on ice for 10 min.</li>
	<li>Add 10 &#181;L of 1% detergent (nonyl phenoxypolyethoxylethanol) to each sample to a final concentration of 0.05%. 
	NOTE: Cells will swell and burst by osmotic lysis as the mild detergent will disrupt the cell membrane.</li>
	<li>Incubate on ice for 5 minutes.</li>
	<li>Vortex briefly and centrifuge at 400 x g at 4 &#730;C for 2 min.</li>
	<li>After vortexing, remove the supernatant and transfer it to a new clean 1.5 mL tube ; this will be the cytoplasmic fraction. Do not discard the pellet.</li>
</ol>
5. Nuclear Isolation
<ol>
	<li>Resuspend the pellet from the previous step with 200 &#181;L of nuclei wash buffer.</li>
	<li>Centrifuge the tube for 2 min at 400 x g and 4 &#176;C.</li>
	<li>Discard the supernatant and repeat the washing procedure two more times. 
	NOTE: The washes will remove cytoplasmic contamination in the nuclear fraction.</li>
	<li>After the third wash, resuspend the pellet in 100 &#181;L of buffer 2.</li>
	<li>Shake the sample vigorously at 4 &#730;C for 30 min. Alternatively, incubate the sample on ice and vortex every 5 min.</li>
	<li>After shaking, centrifuge the sample for 10 min at top speed (&#62; 12,000 x g) and 4 &#176;C.</li>
	<li>After vortexing, transfer the supernatant into a new clean 1.5. mL tube. This will be the nuclear fraction.</li>
</ol>
6. Protein Quantification
Note: Quantify the proteins with a bicinchoninic acid (BCA) protein assay kit. The concentration of proteins in the fractions will range between 0.5 and 5 &#181;g/&#181;L, being lower in the nuclear fraction (see Table 2 for protein concentrations resultant from 2 mg biopsies).
<ol>
	<li>Prepare a standard curve with the following bovine serum albumin (BSA) amounts (&#181;g)12: 0-0.5-1-2-3-4-5-6.</li>
	<li>Prepare the reagents needed to use 100 &#181;L of final BCA mix per sample.</li>
	<li>Pipette 2 &#181;L of each protein fraction in a 96 well plate, add the BCA mix and follow the manufacturer&#39;s instructions for the reading.</li>
</ol>

<ol>
	<li>Cox, B., Emili, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+subcellular+fractionation+and+protein+extraction+for+use+in+mass-spectrometry-based+proteomics.">Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics.</a> Nature Protocols. 1 (4), 1872-1878 (2006).</li><li>Itzhak, D. N., Tyanova, S., Cox, J., Borner, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global,+quantitative+and+dynamic+mapping+of+protein+subcellular+localization.">Global, quantitative and dynamic mapping of protein subcellular localization.</a> eLife. 5, (2016).</li><li>Preedy, V. R., Watson, R. R., Ronald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in disease investigating the gastrointestinal tract. , (1998).</li><li>Alex, P., Gucek, M., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+proteomics+in+the+study+of+inflammatory+bowel+diseases:+Current+status+and+future+directions+with+available+technologies.">Applications of proteomics in the study of inflammatory bowel diseases: Current status and future directions with available technologies.</a> Inflammatory bowel diseases. 15 (4), 616-629 (2009).</li><li>Ericsson, C., Franz&#233;n, B., Nist&#233;r, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frozen+tissue+biobanks.+Tissue+handling,+cryopreservation,+extraction,+and+use+for+proteomic+analysis.">Frozen tissue biobanks. Tissue handling, cryopreservation, extraction, and use for proteomic analysis.</a> Acta oncologica (Stockholm, Sweden). 45 (6), 643-661 (2006).</li><li>Hoffmann, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+application+of+a+subcellular+fractionation+method+to+kidney+and+testis+for+the+determination+of+conjugated+linoleic+acid+in+selected+cell+organelles+of+healthy+and+cancerous+human+tissues.">New application of a subcellular fractionation method to kidney and testis for the determination of conjugated linoleic acid in selected cell organelles of healthy and cancerous human tissues.</a> Analytical and Bioanalytical Chemistry. 381 (6), 1138-1144 (2005).</li><li>Foster, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mammalian+Organelle+Map+by+Protein+Correlation+Profiling.">A Mammalian Organelle Map by Protein Correlation Profiling.</a> Cell. 125 (1), 187-199 (2006).</li><li>Baghirova, S., Hughes, B. G., Hendzel, M. J., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+fractionation+and+isolation+of+subcellular+proteins+from+tissue+or+cultured+cells.">Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells.</a> MethodsX. 2, e440-e445 (2015).</li><li>B&#246;rner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+protein+extraction+from+human+pancreatic+cancer+tissues.">Subcellular protein extraction from human pancreatic cancer tissues.</a> BioTechniques. 46 (4), 297-304 (2009).</li><li>Antalis, T. M., Shea-Donohue, T., Vogel, S. N., Sears, C., Fasano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+disease:+protease+functions+in+intestinal+mucosal+pathobiology.">Mechanisms of disease: protease functions in intestinal mucosal pathobiology.</a> Nature clinical practice. Gastroenterology. 4 (7), 393-402 (2007).</li><li>Cutler, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein purification protocols. , (2004).</li><li>Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bicinchoninic+Acid+(BCA)+Assay+for+Protein+Quantitation.">The Bicinchoninic Acid (BCA) Assay for Protein Quantitation.</a> The Protein Protocols Handbook. , 11-14 (1996).</li><li>McLane, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+localization+of+T-bet+and+Eomes+in+CD8+T+cell+memory+populations.">Differential localization of T-bet and Eomes in CD8 T cell memory populations.</a> Journal of immunology (Baltimore, Md.: 1950). 190 (7), 3207-3215 (2013).</li><li>Nguyen, K. T., Holloway, M. P., Altura, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRM1+nuclear+export+protein+in+normal+development+and+disease.">The CRM1 nuclear export protein in normal development and disease.</a> International journal of biochemistry and molecular biology. 3 (2), 137-151 (2012).</li></ol>

The authors have nothing to disclose.

The protocol described here is used for the nuclear and cytoplasmic fractionation of intestinal biopsies. The purified proteins are not denatured and can be used not only in western blot analysis as shown in Figure&#160;2 and 3 but also in assays requiring native-folded proteins such as immunoprecipitation, electrophoretic mobility shift assay (EMSA) or native polyacrylamide gel electrophoresis (PAGE).
The described method relies on mechanical homogenization of the tissue together with osmotic lysis to burst the cell membrane without affecting the nuclear membrane. The result is a clean fractionation of both the cytoplasmic and nuclear proteins to be used for downstream analyses. It is important to control the degradation of the proteins during the process by the addition of protein and phosphatase inhibitors. Moreover, if extracted proteins are to be used for downstream functional assays, it is also important to avoid denaturation and proteolysis of the samples. The processes should be performed at 4 &#176;C and PMSF should be also added to the buffers.
The procedure described in this protocol is simple and affordable, and the sample processing time is minimal. Problems such as long processing times and the use of costly fractionation kits are not constraints in this protocol.
Fresh and frozen biopsies can both be used in this protocol, with frozen biopsies giving quite clean and reproducible fractionation results as seen in Figure&#160;2 and 3. If there is compartment contamination, the cell lysis time can be reduced to avoid the release of nuclear to the cytoplasm and washing steps can be added to avoid cytoplasmic contamination in the nuclear compartment.
The possibility of using frozen tissues for the independent analysis for nuclear and cytoplasmic protein fractions can increase our knowledge about intestinal inflammatory diseases in which frozen biopsies are stored. Moreover, this technique could be adapted to other kinds of human tissues acquired by biopsies. The volume of buffers used must be modified depending on the size of the biopsy.
The protocol described here is designed to generate reproducible tissue fractions; however, some intrinsic variability within the sample, including the size and the status of the tissue can lead to deviations in the protein amount and distribution managed in each fraction.

Proteins participate in almost all biological functions within a cell and any variation in their structure, quantity or location can lead to a pathogenic scenario. Tissue sample fractionation methods are a useful approach to reduce the complexity of disease related protein analyses. Some studies use protein localization information or enrich a protein from a specific cellular compartment, so a protocol for reproducible fractionation of intact proteins is useful to answer certain biological questions. Determining the subcellular localization of proteins and monitoring their compartmental redistribution or interactions at basal and disease conditions will help identify disease related functional differences1,2. Thus, this method aims to reproducibly fractionate intestinal tissue biopsies, both fresh and frozen, into cytoplasmic and nuclear subcellular compartments.
Intestinal biopsy samples are routinely obtained during endoscopy procedures3 (Figure 1) and can be effectively used for protein quantification or immunoprecipitation studies. Since tissue samples from intestinal epithelial will contain proteins that may be directly involved in the disease pathogenesis4, intestinal biopsy samples are a very valuable source for the successful identification of intestinal disease-specific proteins. Stored frozen, patient tissue samples together with the clinical information are useful resources for protein analysis, and a simple and reproducible sample preparation is a key issue to provide cell compartmental information using limiting amounts of frozen tissue5.
There are several commercially available kits for the separation of cellular fractions, but these are more expensive and generally more time consuming than the protocol presented here. Protocols based on multiple-step gradient centrifugation and continuous gradients have also been previously used for the fractionation of diverse cellular compartments in different tissues6,7. However, maintaining the consistency of continuous gradients is often a difficult task. Similar protocols based on the sequential lysis of membranes have been previously described but they generally need more than two buffers and the hands on time is longer8 .
When fractionating tissue samples, bear in mind that tissue samples present cell-cell and cell-matrix interactions that are both not present in cultured cells and important when proceeding to protein extraction. The proper breakdown between cells and extracellular matrix without affecting the protein quality will be a critical factor in the intestinal tissue protein isolation9. In this protocol, cell-cell and cell-matrix contacts are first broken, releasing the cells and allowing the buffer to reach the individual cells. To avoid protein-compartment mixing prior to fractionation, the breakdown of the contacts must occur without altering the integrity of the cells or the nuclear membranes.
Due to the enrichment of various proteases in the intestinal mucosa10, it is important to control the protein degradation during extraction. To minimize the potential protein degradation, multiple protease inhibitor must be included in the protein extraction buffers. Additionally, if extracts are going to be used for functional assays, it is essential to avoid the denaturation of proteins or proteolysis as this will cause a loss of protein activity11. For this purpose, the protocol is performed at 4 &#176;C and fresh phenylmethylsulfonyl fluoride (PMSF) is added to the buffers at a final concentration of 0.1 mM just prior to use to further inhibit proteolysis.
The first step in cell fractionation is tissue disruption and cell lysis. As mentioned previously, the objective is to disaggregate the cells and break them open with minimum damage. Intestinal tissue samples must be homogenized, and the cells lysed to achieve maximum breakage of the cell membrane. In this protocol, we use a motorized pestle mixer to break the intestinal tissue which is homogenized within seconds by means of high speed vortexing action. After homogenization, tissue cells are incubated in a hypotonic buffer that will burst the cell membrane but will keep the nuclei intact, followed by addition of a non-denaturing detergent (nonyl phenoxypolyethoxylethanol) and a short vortex to separate nuclei from the cytoplasmic fraction. After the removal of the cytoplasm, the cell nuclei membrane is burst into a hypertonic buffer with shaking.
The method presented here is an appropriate method for the subcellular fractionation of fresh and frozen intestinal biopsies that have been obtained during endoscopic procedures and range between 2 and 10 mg in weight. The protocol is easy and reproducible and could be performed using basic laboratory equipment and reagents in under an hour.

<table><tbody><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H4034-1kg</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9333-1kg</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E9884-100G</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>5588886-1kg</td><td></td></tr><tr><td>DTT</td><td>Sigma Aldrich</td><td>10197777001</td><td></td></tr><tr><td>PMSF</td><td>Thermo Scientific</td><td>36978</td><td></td></tr><tr><td>Proteinase and phosphatase inhibitors</td><td>Thermo Scientific</td><td>A32959</td><td></td></tr><tr><td>NP-40&amp;nbsp;</td><td>Sigma Aldrich</td><td>CA-630</td><td>Detergent</td></tr><tr><td>BCA assay</td><td>Thermo Scientific</td><td>23227</td><td>Protein quantification kit</td></tr><tr><td>Disposable plastic pestles</td><td>Sigma Aldrich</td><td>Z359947-100EA</td><td></td></tr><tr><td>Dounce homogeneizer</td><td>VWR</td><td>431-0100</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5415-R</td><td></td></tr><tr><td>Shacker</td><td>IKA</td><td>MS3 basic</td><td></td></tr></tbody></table>

subcellular fractionation from fresh and frozen gastrointestinal specimens

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the localization analysis of specific proteins or protein complexes in different tissue states (i.e., healthy vs. disease). This method is useful for the fractionation of both fresh and frozen intestinal tissue samples; it is easily accessible for all laboratories and not time consuming.

Figure 1&#160;shows a hematoxylin eosin staining of an intestinal biopsy section with intact villi and crypt structures.
Representative results of nuclear and cytoplasmic fractionation using this protocol are shown in Figures 2 and 3. In the experiment shown in Figure 2, a fresh (Fh) and a frozen (Fz) biopsy were simultaneously fractionated following the protocol presented here and a western blot was performed using 20 &#956;g of each sample. Protein concentrations in each fraction and biopsy are shown in Table 2, and the protein concentrations are not very different between the fresh and the frozen tissue. The yield of protein extracted from the 2 mg biopsies is around 3-4 &#181;g/&#181;L in the cytoplasmic fraction and approximately 1.5 &#181;g/&#181;L in the nuclear fraction. To check the purity of the fractions, HSP90 and tubulin were used as a cytoplasmic controls and HDAC1 and H3 as a nuclear controls13. It can be observed that HSP90 and tubulin are primary located in the cytoplasm and HDAC1 and H3 are located in the nucleus with very minimal mixing between the two fractions. To assess whether the protocol affects cell death, we also blotted for the presence of caspase 3. From Figure 2, both samples present a very minimal staining for caspase 3 confirming that the procedure does not affect the cell death pathway even after flash freezing the biopsies. It was also analyzed if protein modifications are maintained after the procedure, so proteins can be used for downstream functional assays. PhosphoSTAT1 was used for staining as the frozen biopsy used in this fractionation came from an individual with an intestinal inflammatory condition; this is not true of the fresh biopsy. As expected, phosphoSTAT1 is located in the cytoplasm of the frozen biopsy, confirming that the protein modifications are maintained after fractionation.
The reproducibility of the method using frozen biopsies is shown in Figure 3. A western blot was performed after fractionation of three independent frozen biopsies, and the fractions are clean with minimal compartment protein mixing (Figure 3A): HSP90 located in the cytoplasmic fraction and HDCA1 in the nuclear fraction. Additionally, the XPO1 protein that is present in both nuclear and cytoplasmic fractions14 was also blotted. It can be observed that the levels of XPO1 quantified by ImageJ are stable in each fraction (Figure 3B), confirming reproducibility of the results using different biopsies.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57740/57740fig1.jpg" /> 
Figure 1: Light micrograph of an intestinal epithelial section, stained with hematoxylin eosin, from a biopsy acquired by endoscopic procedure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57740/57740fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57740/57740fig2.jpg" /> 
Figure 2:&#160;Representative Western blot of a subcellular fractionation performed in a fresh (Fh) and a frozen (Fz) biopsy. HSP90 and tubulin were used as&#160;cytoplasmic controls and HDAC1 and H3&#160;as nuclear controls. The presence of CASP3 and phosphoSTAT3 were&#160;also assessed.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57740/57740fig3v2.jpg" /> 
Figure 3: Fractionation results of three independent frozen biopsies. (A) Representative Western blot of a subcellular fractionation performed in three frozen biopsies (1, 2 and 3). HSP90 was used as a cytoplasmic control and HDAC1 as a nuclear control. The localization of XPO1 was also assessed.&#160;(B)&#160;Relative quantification of XPO1 in each of the fraction done using ImageJ software.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57740/57740fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer</td>
			<td>Components</td>
			<td>Notes</td>
		</tr>
		<tr>
			<td>Hypotonic buffer 1</td>
			<td>10 mM HEPES pH 7.9</td>
			<td>1.5 ml buffer/sample</td>
		</tr>
		<tr>
			<td></td>
			<td>10 mM KCl</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>0.1&#160; nmM EDTA</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypertonic buffer 2</td>
			<td>20 mM HEPES pH 7.9</td>
			<td>150 &#181;l buffer/sample</td>
		</tr>
		<tr>
			<td></td>
			<td>400 mM NaCl</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>0.1&#160; mM EDTA</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">*Note: Buffers can be stored at 4&#186;C for a month.&#160;</td>
		</tr>
		<tr>
			<td colspan="3">**Note: Buffers have to be supplemented with proteinase and phosphatase inhibitors and DTT before use</td>
		</tr>
	</tbody>
</table>
Table 1: Buffer composition.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Fresh</td>
			<td>Frozen</td>
		</tr>
		<tr>
			<td>Cytoplasm</td>
			<td>2.975</td>
			<td>4.612</td>
		</tr>
		<tr>
			<td>Nucleus</td>
			<td>1.516</td>
			<td>1.385</td>
		</tr>
	</tbody>
</table>
Table 2: Protein concentration of each of the fractions in a fresh and frozen 2 mg biopsy used in Figure 2; concentration is presented in &#956;g/&#956;L.

Watch this Scientific Journal Video about Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens at JoVE.com

Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

Here, we present a protocol to perform a simple cellular fractionation for the subcellular separation of cytoplasmic and nuclear proteins in human fresh and frozen intestinal biopsies.

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the ...

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An Overview of the Gastrointestinal System: Anatomy, Physiology, and Assessment

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9.7K Views.  University of the Basque Country (UPV-EHU), Biocruces Health Research Institute. Hier presenteren we een protocol voor het uitvoeren van een eenvoudige cellulaire fractionering voor de subcellular scheiding van cytoplasmatische en nucleaire eiwitten in menselijke verse en diepgevroren intestinale biopsieën.

Video: Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, &#945;-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

Developmental Biology

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Biochemistry

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused on bulk approaches that led to the molecular classification of brain tumors. Over the last five years, single cell sequencing approaches have highlighted several important features of gliomas. The majority of these studies have utilized fresh brain tumor specimens to isolate single cells using flow cytometry or antibody-based separation methods. Moving forward, the use of fresh-frozen tissue samples from biobanks will provide greater flexibility to single cell applications. Furthermore, as the single-cell field advances, the next challenge will be to generate multi-omics data from either a single cell or the same sample preparation to better unravel tumor complexity. Therefore, simple and flexible protocols that allow data generation for various methods such as single-nucleus RNA sequencing (snRNA-seq) and single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq) will be important for the field.
Recent advances in the single cell field coupled with accessible microfluidic instruments such as the 10x genomics platform have facilitated single cell applications in research laboratories. To study brain tumor heterogeneity, we developed an enhanced protocol for the isolation of single nuclei from fresh frozen gliomas. This protocol merges existing single cell protocols and combines a homogenization step followed by filtration and buffer mediated gradient centrifugation. The resulting samples are pure single nuclei suspensions that can be used to generate single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.

Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies.

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

Cancer Research

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

Isolation of Nuclei for Multiome Sequencing in Solid Tumor Specimen

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. 
Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. 
Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

Author Spotlight: Enhancing Nuclei Isolation for Multiome Sequencing in Challenging Tumor Microenvironments

The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei.

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Biology

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Neuroscience

Murine Model for Studying Postoperative Ileus through Intestinal Manipulation

Postoperative Ileus Murine Model

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI is a consequence of mechanical damage during surgery, resulting in disruption of motility in the gastrointestinal tract. The mechanisms of POI are related to aberrant neuronal sensitivity, impaired epithelial barrier function, and increased local inflammation. However, the details remain enigmatic. Therefore, experimental murine models are crucial for elucidating the pathophysiology and mechanism of POI injury and for the development of novel therapies.
Here, we introduce a murine model of POI generated via intestinal manipulation (IM) that is similar to clinical surgery; this is achieved by mechanical damage to the small intestine by massaging the abdomen 1-3 times with a cotton swab. IM delayed gastrointestinal transit 24 h after surgery, as assessed by FITC-dextran gavage and fluorescence detection of the segmental digestive tract. Moreover, tissue swelling of the submucosa and immune cell infiltration were investigated by hematoxylin and eosin staining and flow cytometry. Proper pressure of the IM and a hyperemic effect on the intestine are critical for the procedure. This murine model of POI can be utilized to study the mechanisms of intestinal damage and recovery after abdominal surgery.

We describe a murine model of postoperative ileus generated via intestinal manipulation. Gastrointestinal transit function, pathologic changes, and immune cell activation were assessed 24 h after surgery.

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

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