Name: cell fractionation of u937 cells in the absence of high-speed centrifugation
Uploaded: 2019-01-18T15:00:00
Description: Watch this Scientific Journal Video about Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation at JoVE.com

Work was supported by NIH-1R15HL135675-01 to Timothy J. LaRocca

1. Prepare Buffers and Reagents
NOTE: See Table 1.
<ol>
	<li>Prepare solutions of buffer A, lysis buffer B, sample buffer and digitonin.
	<ol>
		<li>Prepare buffer A by adding 8.77 g of NaCl and 50 mL of HEPES (1 M, pH 7.4) to 900 mL deionized water, adjust final volume to 1 L with deionized water. 
		NOTE: Final concentrations are 150 mM NaCl and 50 mM HEPES.</li>
		<li>Prepare lysis buffer B by adding 20 mL of HEPES (1 M, pH 7.4), 0.75 g of KCl, 0.19 g of MgCl2</...

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The development of this protocol arose from an inability to separate mitochondrial and membrane samples, using commercially available kits, for analysis of protein localization during necroptosis14. The primary limitations of premade kits are their inability to be adapted to the needs of individual researchers, their cost per sample and limited number of samples able to be processed. The method presented here can be performed without the use of expensive reagents and without the necessity for expe...

Reliable identification of protein localization is often necessary when examining molecular pathways in eukaryotic cells. Methods to obtain subcellular fractions are utilized by researchers to more closely examine cellular components of interest.
The majority of existing cell fractionation methods generally fall into two broad categories, detergent-based1,2 and ultracentrifugation-based3,4,5, which can be differentiated by speed, precision and cost. Detergent based protocols rely on the use of buffers with increasing detergent strength to solubilize distinct components of the cell. This is a rapid and convenient method for processing samples and can be cost effective if the number and size of samples are small. Detergent-based kits can be purchased to isolate cytoplasmic, membrane/organelle (mixed fraction), and nuclear fractions from cells. However, several drawbacks associated with these kits limit their usefulness to researchers. They are designed to easily isolate one or two components of the cell, but are incapable of isolating all fractions from a sample concurrently. The use of detergents means that the plasma membrane and membrane-enclosed organelles will be equally solubilized and, therefore, unable to be separated from one another. An additional complication arises from the proprietary components in these kits which prevents researchers from altering conditions for specific applications. Lastly, they are limited in number of uses and may be prohibitively expensive for larger scale experiments. Non-detergent based kits exist for the isolation of mitochondria, however, they are not designed to isolate plasma membrane and the sample yield is significantly less than that from density centrifugation based isolation protocols6,7.
Methods that utilize ultracentrifugation to obtain fractions are more time consuming, but often result in purer fractions than detergent-based kits. To isolate plasma membranes from cells without first solubilizing them (resulting in contamination with membrane organelles) requires them to be lysed by a non-detergent method followed by separation of cellular components via differential centrifugation&#8212;with plasma membrane isolation requiring speeds of 100,000 &#215; g to accomplish. In many cases, differential centrifugation must be followed by isopycnic density gradient centrifugation for further separation of cellular fractions or removal of contaminants. While these methods are thorough and modifiable, drawbacks include cost, time consumption, and the need for an ultracentrifuge for separation of fractions and further purification via density gradient centrifugation. Most high-speed centrifuges are at a cost that is prohibitive for individual investigators and are often shared, core equipment at academic institutions. Thus, ultracentrifuge availability becomes prohibitive in these situations.
In this fractionation protocol we demonstrate the isolation of subcellular fractions without the use of solubilizing detergents and without high speed centrifugation. This method will allow researchers to isolate the plasma membrane, mitochondria and cytoplasmic components of a eukaryotic cell with minimal contamination between fractions.

<table>
	<tbody>
		<tr>
			<td>Digitonin</td>
			<td>TCI Chemicals</td>
			<td>D0540</td>
			<td>For Cytoplasm Extraction</td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>M4125</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Dounce homogenizer</td>
			<td>VWR</td>
			<td>22877-282</td>
			<td>For Homogenization</td>
		</tr>
		<tr>
			<td>end-over-end rotator</td>
			<td>Barnstead</td>
			<td>N/A</td>
			<td>For Cytoplasm Extraction</td>
		</tr>
		<tr>
			<td>ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td>Alfa Aesar</td>
			<td>J61721</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>GAPDH (14C10)</td>
			<td>Cell Signalling Technologies</td>
			<td>2118</td>
			<td>For detection of cytoplasmic fractions on western blot, dilution: 1:10000</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>VWR</td>
			<td>J848</td>
			<td>For Lysis buffers A and B</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Alfa Aesar</td>
			<td>12315</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Na, K-ATPase a1 (D4Y7E)</td>
			<td>Cell Signalling Technologies</td>
			<td>23565</td>
			<td>For detection of plasma membrane fractions on western blot, dilution: 1:1000</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>793566</td>
			<td>For Lysis buffer A</td>
		</tr>
		<tr>
			<td>phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>VWR</td>
			<td>M145</td>
			<td>For Cytoplasm Extraction and Homogenization Buffer</td>
		</tr>
		<tr>
			<td>probe sonicator</td>
			<td>Qsonica</td>
			<td>Q125-110</td>
			<td>For Final Samples</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail, General Use</td>
			<td>VWR</td>
			<td>M221-1ML</td>
			<td>For Cytoplasm Extraction</td>
		</tr>
		<tr>
			<td>refrigerated centrifuge</td>
			<td>Beckman-Coulter</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>VWR</td>
			<td>227</td>
			<td>For Sample buffer</td>
		</tr>
		<tr>
			<td>sodium orthovanadate (SOV)</td>
			<td>Sigma-Aldrich</td>
			<td>450243</td>
			<td>For Lysis buffers A and B</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Tris-buffered Saline (TBS)</td>
			<td>VWR</td>
			<td>788</td>
			<td>For Sample buffer</td>
		</tr>
		<tr>
			<td>VDAC (D73D12)</td>
			<td>Cell Signalling Technologies</td>
			<td>4661</td>
			<td>For detection of mitochondrial fractions on western blot, dilution: 1:1000</td>
		</tr>
	</tbody>
</table>

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.

Successful fractionation of undifferentiated U9378 cells grown in suspension was accomplished using the protocol detailed above and illustrated in Figure 1. The samples obtained with this method were subjected to western blotting9 utilizing a wet transfer method to a polyvinylidene fluoride (PVDF) membrane. The membrane was subsequently probed with antibodies against cytoplasmic, mitochondrial and membrane local...

Watch this Scientific Journal Video about Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation at JoVE.com

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

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. ...

This protocol allows researchers to obtain distinct fractions of a cell without the use of specialized equipment, expensive reagents or complex techniques.The primary advantage of this protocol is the simplicity of the techniques which make it modifiable and easy to perform.Demonstrating the procedure will be Matt Deragon, Rob Haluska and Alexa Hodges.Three graduate students from my laboratory.To begin centrifuge the prepared cell suspension to create a pellet.Remove the supernatant and re-suspend the pellet in room temperature PBS.Centrifuge the cell suspension at 400 xg for ten minutes.Then remove the supernatant and re-suspend the cell pellet in ice cold buffer.Prepare buffer A solution according to the text protocol.Centrifuge the cell suspension at 400 xg for ten minutes and remove the supernatant.Re-suspend the pellet in buffer A solution to a final concentration of 200 million cells per ml, and pipette gently to break up any clumps.After this incubate the cell suspension on an end over end rotator at 4

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