Name: cell fractionation of u937 cells by isopycnic density gradient purification
Uploaded: 2021-08-12T13:00:00
Description: Watch this Scientific Journal Video about Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification at JoVE.com

This work was supported by NIH R15-HL135675-01 and NIH 2 R15-HL135675-02 to T.J.L.

1. Prepare buffers and reagents
<ol>
	<li>Prepare fresh solutions of phosphatase and protease inhibitors.
	<ol>
		<li>Add 17.4 mg of phenylmethanesulfonyl fluoride (PMSF)to 1 mL of 100% ethanol to prepare a 100 mM stock. 
		&#8203;NOTE: Wear protective equipment when handling PMSF as it is hazardous when ingested or inhaled and upon contact with skin or eyes. It is corrosive to eyes and skin.</li>
		<li>According to the manufacturer&#39;s instructions, prepare a commercially available protease inhibitor cocktail (...

<ol>
	<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, 440-445 (2015).</li><li>Il Hwang, S., Han, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+fractionation+for+identification+of+biomarkers:+Serial+detergent+extraction+by+subcellular+accessibility+and+solubility.">Subcellular fractionation for identification of biomarkers: Serial detergent extraction by subcellular accessibility and solubility.</a> Methods in Molecular Biology. 1002, 25-35 (2013).</li><li>Clayton, D. A., Shadel, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondria+from+cells+and+tissues.">Isolation of mitochondria from cells and tissues.</a> Cold Spring Harbor Protocols. 2014 (10), 1040-1041 (2014).</li><li>Stimpson, S. E., Coorssen, J. R., Myers, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+isolation+of+mitochondria+for+proteomic+analyses.">Optimal isolation of mitochondria for proteomic analyses.</a> Analytical Biochemistry. 475, 1-3 (2015).</li><li>Sundstr&#246;m, C., Nilsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+histiocytic+lymphoma+cell+line+(U-937).">Establishment and characterization of a human histiocytic lymphoma cell line (U-937).</a> International Journal of Cancer. 17 (5), 565-577 (1976).</li><li>Hodge, T., Colombini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+metabolite+flux+through+voltage-gating+of+VDAC+channels.">Regulation of metabolite flux through voltage-gating of VDAC channels.</a> Journal of Membrane Biology. 157 (3), 271-279 (1997).</li><li>Therien, A. G., Blostein, R. <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+sodium+pump+regulation.+American+journal+of+physiology.">Mechanisms of sodium pump regulation. American journal of physiology.</a> Cell physiology. 279 (3), 541-566 (2000).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proceedings of the National Academy of Sciences of the United States of the America. 76 (9), 4350-4354 (1979).</li><li>Barber, R. D., Harmer, D. W., Coleman, R. A., Clark, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAPDH+as+a+housekeeping+gene:+analysis+of+GAPDH+mRNA+expression+in+a+panel+of+72+human+tissues.">GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues.</a> Physiological Genomics. 21 (3), 389-395 (2005).</li><li>Bradbury, E. M., Cary, P. D., Crane-Robinson, C., Rattle, H. W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformations+and+interactions+of+histones+and+their+role+in+chromosome+structure.">Conformations and interactions of histones and their role in chromosome structure.</a> Annals of the New York Academy of Sciences. 222, 266-289 (1973).</li><li>McCaig, W. D., Deragon, M. A., Haluska, R. J., Hodges, A. L., Patel, P. S., LaRocca, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fractionation+of+U937+cells+in+the+absence+of+high-speed+centrifugation.">Cell fractionation of U937 cells in the absence of high-speed centrifugation.</a> Journal of Visualized Experiments. (143), e59022 (2019).</li><li>McCaig, W. D., 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=Hyperglycemia+potentiates+a+shift+from+apoptosis+to+RIP1-dependent+necroptosis.">Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis.</a> Cell Death Discovery. 4, 55 (2018).</li></ol>

This method is a modified version of a previously published approach to subcellular fractionation without the use of high-speed centrifugation11. This modified method requires more specialized equipment to achieve the best results, but is more comprehensive and consistently reproducible.
The development of the initial protocol was necessary due to an inability to separate mitochondrial and membrane samples for the analysis of protein localization during necroptosis<sup ...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62119">Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification</a>. The Authors section was&#160;updated.
The Authors section was updated from:
William McCaig1 
Timothy LaRocca1 
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences
to:
William D. McCaig1 
Matthew A. Deragon1 
Phillip V. Truong1 
Angeleigh R. Knapp1 
Keven J. Hughes1 
Timothy J. LaRocca1 
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62119">Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification</a>. The Authors section was&#160;updated.

Erratum: Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62119">Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification</a>. The Authors section was&#160;updated.
The Authors section was updated from:
William D. McCaig1 
Matthew A. Deragon1 
Phillip V. Truong1 
Angeleigh R. Knapp1 
Keven J. Hughes1 
Timothy J. LaRocca1 
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences
to:
William D. McCaig1 
Matthew A. Deragon1 
Phillip V. Truong1 
Angeleigh R. Knapp1 
Timothy J. LaRocca1 
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences

Subcellular fractionation is a procedure in which cells are lysed and separated into their constituent components through several methods. This technique can be used by researchers to determine protein localization in mammalian cells or for enrichment of low-abundance proteins that would otherwise be undetectable. While methods for subcellular fractionation currently exist, as do commercial kits that can be purchased, they suffer from several limitations that this procedure attempts to overcome. Most cell fractionation methods are exclusively detergent-based1,2, relying on the use of buffers containing increasing amounts of detergent to solubilize different cellular components. While this method is rapid and convenient, it results in impure fractions. These are designed to allow researchers to easily isolate one or two components of the cell, but are not complex enough to isolate multiple subcellular fractions from a sample at the same time. Relying solely on detergents usually results in membrane-enclosed organelles and the plasma membrane being indiscriminately solubilized, making separation of these components difficult. An additional complication from the use of these kits is the inability of researchers to alter/optimize them for specific applications, as most of the components are proprietary formulations. Finally, these kits can be prohibitively expensive, with limitations in the number of uses that make them less than ideal for larger samples. 
Despite the availability of kits for the isolation of mitochondria that do not rely on detergents, they are not designed to isolate the plasma membrane and yield significantly lower amounts of sample than standard isolation protocols3,4. While differential centrifugation methods are more time-consuming, they often result in distinct fractions that cannot be obtained with exclusively detergent-based kits1. Separation without the sole use of solubilizing detergents also allows further purification using ultracentrifugation and isopycnic density gradients, resulting in less cross-contamination. This fractionation protocol demonstrates the isolation of subcellular fractions from U937 monocytes using a combination of detergent- and high-speed centrifugation-based approaches. This method will facilitate the isolation of the nuclear, cytoplasmic, mitochondrial, and plasma membrane components of a mammalian cell with minimal contamination between the fractions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Bullet Blender Tissue Homogenizer</td>
			<td>Next Advance</td>
			<td>61-BB50-DX</td>
			<td></td>
		</tr>
		<tr>
			<td>digitonin</td>
			<td>Sigma</td>
			<td>D141</td>
			<td></td>
		</tr>
		<tr>
			<td>end-over-end rotator</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH (14C10)&#160;</td>
			<td>Cell Signalling Technologies</td>
			<td>2118</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>VWR</td>
			<td>97064-360</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexylene glycol</td>
			<td>Sigma</td>
			<td>68340</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal</td>
			<td>Sigma</td>
			<td>I7771</td>
			<td>Non-ionic, non-denaturing detergent</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma</td>
			<td>M9647</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Na, K-ATPase a1 (D4Y7E)</td>
			<td>Cell Signalling Technologies</td>
			<td>23565</td>
			<td></td>
		</tr>
		<tr>
			<td>Open-Top Polyclear Tubes, 16 x 52 mm</td>
			<td>Seton Scientific</td>
			<td>7048</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep (Iodixanol)&#160;Density Gradient Medium</td>
			<td>Sigma</td>
			<td>D1556-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail, General Use</td>
			<td>VWR</td>
			<td>M221-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>refrigerated centrifuge</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S50-ST Swinging Bucket Rotor</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma</td>
			<td>436143</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium orthovanadate (SOV)</td>
			<td>Sigma</td>
			<td>567540</td>
			<td></td>
		</tr>
		<tr>
			<td>sonicator</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall MX120 Plus Micro-Ultracentrifuge</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Beads 3.2 mm</td>
			<td>Next Advance</td>
			<td>SSB32</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered Saline (TBS)</td>
			<td>VWR</td>
			<td>97062-370</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td></td>
			<td></td>
			<td>non-ionic detergent in western blotting buffers</td>
		</tr>
		<tr>
			<td>VDAC (D73D12)</td>
			<td>Cell Signalling Technologies</td>
			<td>4661</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A schematic flow chart of this procedure (Figure 1) visually summarizes the steps that were taken to successfully fractionate U9375 cells grown in suspension. Fractions collected from the top of the isopycnic density gradient in equal volumes (1 mL) show the purification of the mitochondrial and membrane fractions (Figure 2). Utilizing an antibody against VDAC, a protein localized to the outer mitochondrial membrane6</su...

Watch this Scientific Journal Video about Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification at JoVE.com

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

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 ...

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