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, 2 mL of Ethylenediaminetetraacetic acid (0.5 M EDTA), 2 mL of ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (0.5 M EGTA), 38.26 g of mannitol and 23.96 g of sucrose to 900 mL of deionized water, adjust final volume to 1 L with deionized water. 
		NOTE: Final concentrations are 20 mM HEPES, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 210 mM mannitol and 70 mM sucrose.</li>
		<li>Prepare sample buffer by adding 0.01 g of sodium dodecyl sulfate (SDS) to 10 mL of tris-buffered saline (TBS) for a final concentration of 0.1% SDS.</li>
		<li>Prepare a stock solution of digitonin by adding 25 mg of digitonin to 100 mL of deionized water (final concentration is 250 &#181;g/mL).</li>
		<li>Store all buffer solutions at 4 &#176;C and digitonin at -20 &#176;C until the start of experiment.</li>
	</ol>
	</li>
	<li>Prepare fresh solutions of protease and phosphatase inhibitors to be added to buffer solutions prior to addition to cells.
	<ol>
		<li>Prepare a stock solution of phenylmethanesulfonyl fluoride (PMSF) by adding 17.4 mg of PMSF to 1 mL of 100% ethanol (final concentration is 100 mM). 
		CAUTION: Wear appropriate protective equipment and exercise caution when handling PMSF. PMSF is hazardous if ingested and slightly hazardous in case of skin contact (irritant), eye contact (irritant) or inhalation; it is corrosive to eyes and skin.</li>
		<li>Prepare a commercially available protease inhibitor cocktail (100&#215;) according to the manufacturer&#8217;s instructions (see the Table of Materials).</li>
		<li>Prepare a stock solution of sodium orthovanadate (SOV) by adding 92 mg of SOV to 1 mL of deionized water (final concentration is 500 mM). 
		CAUTION: Wear appropriate protective equipment and use caution when handling. SOV is hazardous in case of eye contact (irritant), ingestion or inhalation. Severe over-exposure can result in death.</li>
	</ol>
	</li>
</ol>
2. PBS Wash
<ol>
	<li>Concentrate and wash cells in phosphate-buffered saline (PBS) prior to fractionation.
	<ol>
		<li>Centrifuge cell suspension at an appropriate speed to create a pellet. For example, centrifuge a suspension of U937 cells at 400 &#215; g for 10 min.</li>
		<li>Remove the supernatant, resuspend cell pellet in room-temperature PBS at a final concentration of 4 &#215; 106 cells/mL and pipette gently to break up clumps.</li>
		<li>Centrifuge the cell suspension at 400 &#215; g for 10 min to pellet cells.</li>
		<li>Remove the supernatant and resuspend cell pellet in ice cold buffer A at a final concentration of 2 &#215; 107 cells/mL. 
		NOTE: All subsequent steps should be carried out at 4 &#176;C or on ice and all buffers should be pre-chilled.</li>
	</ol>
	</li>
</ol>
3. Cytosolic Protein Isolation
<ol>
	<li>Extract cytosolic proteins by incubation with the detergent digitonin.
	<ol>
		<li>Immediately prior to resuspension of cells (step 3.1.3) add 10 &#181;L of stock PMSF (100 mM), 10 &#181;L of protease Inhibitor (100&#215;), 2 &#181;L of stock SOV (500 mM) and 100 &#181;L of stock digitonin (250 &#181;g/mL) to 878 &#181;L of buffer A (final concentrations are 1 mM PMSF, 1&#215; Protease Inhibitor, 1 mM SOV and 25 &#181;g/mL digitonin; adjust the final volume as per the number of cells being used). Keep the solution on ice until addition to cell pellet.</li>
		<li>Centrifuge the cell suspension at 400 &#215; g for 10 min and remove the supernatant.</li>
		<li>Resuspend the cell pellet in buffer A solution containing inhibitors and digitonin (prepared in step 3.1.1) at a final concentration of 2 &#215; 107 cells/mL, pipette gently to break up clumps.</li>
		<li>Incubate the cell suspension on an end-over-end rotator at 4 &#176;C for 20 min.</li>
		<li>Centrifuge the cell suspension at 400 &#215; g for 10 min. Collect the supernatant and place it in a clean centrifuge tube.</li>
		<li>Centrifuge the collected supernatant at 18,000 &#215; g for 20 min to pellet cellular debris.</li>
		<li>Transfer the supernatant to a clean centrifuge tube.</li>
		<li>Repeat steps 3.1.5 and 3.1.6 until no pellet is obtained following centrifugation.</li>
		<li>Collect the supernatant containing the cytosolic proteins and store it at 4 &#176;C (short term) or -20 &#176;C (long term).</li>
	</ol>
	</li>
	<li>Remove excess digitonin and cytosolic proteins by centrifugation.
	<ol>
		<li>Resuspend the digitonin-permeabilized cell pellet (from step 3.1.5) in buffer A at a final concentration of 4 &#215; 106 cells/mL and pipette gently to break up clumps.</li>
		<li>Centrifuge the digitonin-permeabilized cell suspension at 400 &#215; g for 10 min and remove the supernatant. 
		NOTE: Repeated washes in buffer A can be performed to remove excess cytosolic contaminants.</li>
	</ol>
	</li>
</ol>
4. Cell Homogenization
<ol>
	<li>Incubate the cells on ice in lysis buffer B and lyse them by mechanical means.
	<ol>
		<li>Immediately prior to resuspension of cells (step 4.1.2) add 10 &#181;L of stock PMSF (100 mM) and 2 &#181;L of stock SOV (500 mM) to 988 &#181;L of lysis buffer B (final concentrations are 1 mM PMSF and 1 mM SOV; adjust final volume to accommodate number of cells being lysed) and keep solution on ice until addition to cell pellet.</li>
		<li>Resuspend the cell pellet (from step 3.2.2) in ice cold lysis buffer B solution containing PMSF and SOV (prepared in step 4.1.1) at a final concentration of 4 &#215; 106 cells/mL.</li>
		<li>Incubate the cell suspension on ice for 30 min.</li>
		<li>Transfer the cell suspension to a pre-chilled Dounce homogenizer (with a tight-fitting B pestle) on ice and perform 40 passes with the homogenizer pestle using slow, even strokes. 
		NOTE: Alternatively utilize other means of mechanical cell lysis as detailed in discussion section.</li>
		<li>Collect the homogenate and transfer it to a clean centrifuge tube.</li>
		<li>Wash the homogenizer pestle and tube with a small volume (1 to 2 mL) of lysis buffer B and add it to the homogenate.</li>
		<li>Centrifuge the homogenate at 400 &#215; g (or the minimum speed required to pellet unbroken cells) for 10 min.</li>
		<li>Transfer the supernatant to a clean centrifuge tube. 
		Note: If a significant pellet remains repeat steps 4.1.4 through 4.1.6 to increase the yield of fractions as detailed in the discussion section. The protocol can be paused here, and the homogenate stored at 4 &#176;C for short term (24 h).</li>
	</ol>
	</li>
</ol>
5. Differential Centrifugation
<ol>
	<li>Centrifuge the homogenate at increasing speeds to remove cellular debris, isolate mitochondria and membrane fractions.
	<ol>
		<li>Centrifuge the supernatant (from step 4.1.8) at 500 &#215; g for 10 min. Transfer supernatant to a clean centrifuge tube, and discard any pellet.</li>
		<li>Centrifuge supernatant (from step 5.1.1) at 1,000 &#215; g for 10 min. Transfer the supernatant to a clean centrifuge tube, and discard any pellet.</li>
		<li>Centrifuge the supernatant (from step 5.1.2) at 2,000 &#215; g for 10 min. Transfer the supernatant to a clean centrifuge tube, and discard any pellet.</li>
		<li>Centrifuge the supernatant (from step 5.1.3) at 4,000 &#215; g for 15 min. Transfer supernatant to a clean centrifuge tube, keep pellet containing mitochondria.</li>
		<li>Resuspend the mitochondria pellet in a small volume (0.5&#8210;1 mL) of lysis buffer B.</li>
		<li>Centrifuge the suspended pellet at 4,000 &#215; g for 15 min. Remove the supernatant and resuspend the mitochondrial pellet in the desired final volume of sample buffer (e.g., 250 to 500 &#181;L, depending on the size of the pellet and desired concentration).</li>
		<li>Centrifuge the supernatant (from step 5.1.4) at 4,000 &#215; g for 15 min. Transfer the supernatant to a clean centrifuge tube. Repeat this step until no pellet is obtained following centrifugation.</li>
		<li>Spin the supernatant at 18,000 &#215; g for 3 h.</li>
		<li>Remove the supernatant, and keep the pellet containing membrane proteins. Resuspend the membrane pellet in a small volume (0.5&#8211;1 mL) of lysis buffer B.</li>
		<li>Centrifuge the suspended pellet at 18,000 &#215; g for 1 h.</li>
		<li>Remove the supernatant and resuspend the membrane pellet in the desired final volume of sample buffer (250 to 500 &#181;L, depending on the size of the pellet and desired concentration).</li>
	</ol>
	</li>
	<li>Sonicate the sample pellets for 3 s in an ice bath at a power setting of 5 (50% of 125 W maximum power at 20 kHz, see the Table of Materials).</li>
	<li>Store the samples at 4 &#176;C (short term) or -20 &#176;C (long term).</li>
	<li>Examine the samples for purity of fractionation by performing a western blot utilizing antibodies against protein markers found in the cytoplasm, mitochondria and membrane compartments of the cell (refer to the representative results section).</li>
</ol>

<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, e440-e445 (2015).</li><li>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>Song, Y., Hao, Y., 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=Sample+preparation+project+for+the+subcellular+proteome+of+mouse+liver.">Sample preparation project for the subcellular proteome of mouse liver.</a> Proteomics. 6 (19), 5269-5277 (2006).</li><li>Lenstra, J. A., Bloemendal, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topography+of+the+total+protein+population+from+cultured+cells+upon+fractionation+by+chemical+extractions.">Topography of the total protein population from cultured cells upon fractionation by chemical extractions.</a> European Journal of Biochemistry. 135 (3), 413-423 (1983).</li><li>Michelsen, U., von Hagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+19+Isolation+of+Subcellular+Organelles+and+Structures.">Chapter 19 Isolation of Subcellular Organelles and Structures.</a> Methods in Enzymology. 463 (C), 305-328 (2009).</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>Williamson, C. D., Wong, D. S., Bozidis, P., Zhang, A., Colberg-Poley, A. M. <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+Endoplasmic+Reticulum,+Mitochondria,+and+Mitochondria-Associated+Membrane+and+Detergent+Resistant+Membrane+Fractions+from+Transfected+Cells+and+from+Human+Cytomegalovirus-Infected+Primary+Fibroblasts.">Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts.</a> Current protocols in cell biology. 68, (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>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. 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>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), C541-C566 (2000).</li><li>Devarajan, P., Stabach, P. R., De Matteis, M. A., Morrow, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na,K-ATPase+transport+from+endoplasmic+reticulum+to+Golgi+requires+the+Golgi+spectrin-ankyrin+G119+skeleton+in+Madin+Darby+canine+kidney+cells.">Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in Madin Darby canine kidney cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 94 (20), 10711-10716 (1997).</li><li>McCaig, W. D., Patel, P. S., 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><li>Simpson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogenization+of+Mammalian+Tissue.">Homogenization of Mammalian Tissue.</a> Cold Spring Harbor Protocols. 2010 (7), (2010).</li></ol>

The authors declare no conflict of interest

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(&amp;beta;-aminoethyl ether)-N,N,N&#039;,N&#039;-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, &lt;u&gt;dilution&lt;/u&gt;: 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>MgCl&lt;sub&gt;2&lt;/sub&gt;</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, &lt;u&gt;dilution&lt;/u&gt;: 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&lt;strong&gt;)&lt;/strong&gt;</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, &lt;u&gt;dilution&lt;/u&gt;: 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. ...

cell-fractionation-u937-cells-absence-high-speed

Research

JoVE Journal

Biochemistry

13.5K Views.  Albany College of Pharmacy and Health Sciences. 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.

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

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Cancer Research

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

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 ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Immunology and Infection

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Biology

Extraction and Identification of Micronuclei from Human Peripheral Blood Lymphocytes

A micronucleus (MN) is an abnormal nuclear structure that forms in cells, particularly bone marrow or blood cells, when exposed to external damage, such as radiation, due to unresolved DNA damage or mitotic errors. Once formed, MNs can actively contribute to various carcinogenic processes, including inflammatory signaling and chromosomal genetic rearrangements. MNs contain nuclear DNA, histones, nucleoprotein fragments, and other active proteins, which are closely associated with their functions. Studying the formation and components of MNs is crucial for understanding their role in driving carcinogenic processes. The extraction and purification of MNs are essential to achieving these research objectives. However, the instability of the MN nuclear membrane and its susceptibility to rupture make these processes technically challenging. Currently, only a few studies have reported the use of density gradient centrifugation for MN separation. This study summarizes and simplifies the processes of MN separation and purification. Human peripheral blood lymphocytes exposed to radiation were isolated, and MNs were separated and purified using sucrose buffers of different concentrations in a two-step process. The integrity and purity of the MNs were verified, providing a clear and practical demonstration of the experimental procedure for researchers investigating the causes and functions of MNs.

This protocol outlines a method for extracting and purifying micronuclei from human lymphocytes using sucrose density gradient centrifugation. It provides an experimental basis for investigating the composition and function of micronuclei.

A micronucleus (MN) is an abnormal nuclear structure that forms in cells, particularly bone marrow or blood cells, when exposed to external damage, such ...

Medicine

Preparation of Subcellular Fractions from Leukemic Cells: A Method to Generate Cellular and Nuclear Fractions from Leukemic Cells

Source: Hay, J., et al. <a href="https://www.jove.com/t/60426">Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking.</a> J. Vis. Exp. (2019).
This video describes the preparation of subcellular fractions from leukemic cells which is a method to generate cellular and nuclear fractions from leukemic cells. The nuclear and cytoplasmic fractions can be used for further downstream analysis.

Przygotowanie frakcji subkomórkowych z komórek białaczkowych: metoda generowania frakcji komórkowych i jądrowych z komórek białaczkowych

Source: Hay, J., et al. Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking. J. ...

JoVE Encyclopedia of Experiments

Leukemia

In vitro techniques & assays

Isolating Tumor-Infiltrating Immune Cells from a Murine Brain

Source: Baker, G. J., et al., Lowenstein, P. R. <a href="https://app.jove.com/v/53676">Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells.</a> J. Vis. Exp. (2015)
The video demonstrates the isolation of tumor-infiltrating peripheral blood mononuclear cells from a mouse brain using mechanical dissociation and enzymatic digestion. This is followed by density gradient centrifugation to collect the immune cells for further analysis.

Izolowanie komórek odpornościowych naciekających nowotwór z mysiego mózgu

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Prepare density centrifugation media ...

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Extraction and Identification of Micronuclei from Human Peripheral Blood Lymphocytes

Przygotowanie frakcji subkomórkowych z komórek białaczkowych: metoda generowania frakcji komórkowych i jądrowych z komórek białaczkowych

Izolowanie komórek odpornościowych naciekających nowotwór z mysiego mózgu