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

Summary

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.

Abstract

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.

Introduction

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-based^1,2 and ultracentrifugation-based^3,4,5, 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 protocols^6,7.

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—with plasma membrane isolation requiring speeds of 100,000 × 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.

Protocol

1. Prepare Buffers and Reagents

NOTE: See Table 1.

1. Prepare solutions of buffer A, lysis buffer B, sample buffer and digitonin.
   1. 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.
   2. 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(β-aminoethyl ether)-N,N,N',N'-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.
   3. 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.
   4. Prepare a stock solution of digitonin by adding 25 mg of digitonin to 100 mL of deionized water (final concentration is 250 µg/mL).
   5. Store all buffer solutions at 4 °C and digitonin at -20 °C until the start of experiment.
2. Prepare fresh solutions of protease and phosphatase inhibitors to be added to buffer solutions prior to addition to cells.
   1. 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.
   2. Prepare a commercially available protease inhibitor cocktail (100×) according to the manufacturer’s instructions (see the Table of Materials).
   3. 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.

2. PBS Wash

1. Concentrate and wash cells in phosphate-buffered saline (PBS) prior to fractionation.
   1. Centrifuge cell suspension at an appropriate speed to create a pellet. For example, centrifuge a suspension of U937 cells at 400 × g for 10 min.
   2. Remove the supernatant, resuspend cell pellet in room-temperature PBS at a final concentration of 4 × 10⁶ cells/mL and pipette gently to break up clumps.
   3. Centrifuge the cell suspension at 400 × g for 10 min to pellet cells.
   4. Remove the supernatant and resuspend cell pellet in ice cold buffer A at a final concentration of 2 × 10⁷ cells/mL.
      NOTE: All subsequent steps should be carried out at 4 °C or on ice and all buffers should be pre-chilled.

3. Cytosolic Protein Isolation

1. Extract cytosolic proteins by incubation with the detergent digitonin.
   1. Immediately prior to resuspension of cells (step 3.1.3) add 10 µL of stock PMSF (100 mM), 10 µL of protease Inhibitor (100×), 2 µL of stock SOV (500 mM) and 100 µL of stock digitonin (250 µg/mL) to 878 µL of buffer A (final concentrations are 1 mM PMSF, 1× Protease Inhibitor, 1 mM SOV and 25 µg/mL digitonin; adjust the final volume as per the number of cells being used). Keep the solution on ice until addition to cell pellet.
   2. Centrifuge the cell suspension at 400 × g for 10 min and remove the supernatant.
   3. Resuspend the cell pellet in buffer A solution containing inhibitors and digitonin (prepared in step 3.1.1) at a final concentration of 2 × 10⁷ cells/mL, pipette gently to break up clumps.
   4. Incubate the cell suspension on an end-over-end rotator at 4 °C for 20 min.
   5. Centrifuge the cell suspension at 400 × g for 10 min. Collect the supernatant and place it in a clean centrifuge tube.
   6. Centrifuge the collected supernatant at 18,000 × g for 20 min to pellet cellular debris.
   7. Transfer the supernatant to a clean centrifuge tube.
   8. Repeat steps 3.1.5 and 3.1.6 until no pellet is obtained following centrifugation.
   9. Collect the supernatant containing the cytosolic proteins and store it at 4 °C (short term) or -20 °C (long term).
2. Remove excess digitonin and cytosolic proteins by centrifugation.
   1. Resuspend the digitonin-permeabilized cell pellet (from step 3.1.5) in buffer A at a final concentration of 4 × 10⁶ cells/mL and pipette gently to break up clumps.
   2. Centrifuge the digitonin-permeabilized cell suspension at 400 × g for 10 min and remove the supernatant.
      NOTE: Repeated washes in buffer A can be performed to remove excess cytosolic contaminants.

4. Cell Homogenization

1. Incubate the cells on ice in lysis buffer B and lyse them by mechanical means.
   1. Immediately prior to resuspension of cells (step 4.1.2) add 10 µL of stock PMSF (100 mM) and 2 µL of stock SOV (500 mM) to 988 µL 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.
   2. 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 × 10⁶ cells/mL.
   3. Incubate the cell suspension on ice for 30 min.
   4. 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.
   5. Collect the homogenate and transfer it to a clean centrifuge tube.
   6. Wash the homogenizer pestle and tube with a small volume (1 to 2 mL) of lysis buffer B and add it to the homogenate.
   7. Centrifuge the homogenate at 400 × g (or the minimum speed required to pellet unbroken cells) for 10 min.
   8. 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 °C for short term (24 h).

5. Differential Centrifugation

1. Centrifuge the homogenate at increasing speeds to remove cellular debris, isolate mitochondria and membrane fractions.
   1. Centrifuge the supernatant (from step 4.1.8) at 500 × g for 10 min. Transfer supernatant to a clean centrifuge tube, and discard any pellet.
   2. Centrifuge supernatant (from step 5.1.1) at 1,000 × g for 10 min. Transfer the supernatant to a clean centrifuge tube, and discard any pellet.
   3. Centrifuge the supernatant (from step 5.1.2) at 2,000 × g for 10 min. Transfer the supernatant to a clean centrifuge tube, and discard any pellet.
   4. Centrifuge the supernatant (from step 5.1.3) at 4,000 × g for 15 min. Transfer supernatant to a clean centrifuge tube, keep pellet containing mitochondria.
   5. Resuspend the mitochondria pellet in a small volume (0.5‒1 mL) of lysis buffer B.
   6. Centrifuge the suspended pellet at 4,000 × 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 µL, depending on the size of the pellet and desired concentration).
   7. Centrifuge the supernatant (from step 5.1.4) at 4,000 × g for 15 min. Transfer the supernatant to a clean centrifuge tube. Repeat this step until no pellet is obtained following centrifugation.
   8. Spin the supernatant at 18,000 × g for 3 h.
   9. Remove the supernatant, and keep the pellet containing membrane proteins. Resuspend the membrane pellet in a small volume (0.5–1 mL) of lysis buffer B.
   10. Centrifuge the suspended pellet at 18,000 × g for 1 h.
   11. Remove the supernatant and resuspend the membrane pellet in the desired final volume of sample buffer (250 to 500 µL, depending on the size of the pellet and desired concentration).
2. 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).
3. Store the samples at 4 °C (short term) or -20 °C (long term).
4. 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).

Results

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...

Discussion

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...

Materials

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