This work was supported by NIH CA109821 (to TCC). We thank Beckman-Coulter for generously providing funds to cover publication costs and for technical assistance in the set-up and operation of the elutriation system. We thank Dr. Fred Falkenburg for providing initial primary ALL cultures.

NOTE: This protocol has been optimized for primary B-ALL cells which range in diameter from approximately 8 &#181;m (G1 phase) to 13 &#181;m (G2/M phases). Thus, the specific centrifuge speeds and pump flow rates may not be applicable to cells with different size ranges. Nevertheless, appropriate elutriation parameters can be estimated using the sedimentation rate equation. Cells are cultured in a defined serum-free medium as described16 at an optimal density of 1 - 3 x 106 cells...

<ol>
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Anisha Kothari is currently at&#160;the Department of Cell and Molecular Biology at St. Jude Children&#39;s Research Hospital.

We have described a method for obtaining primary ALL cells in different phases of the cell cycle using CCE. Under optimal conditions an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases could readily be obtained in excellent yield, and cells highly enriched in S phase can also be obtained if desired. The results presented here were obtained using one primary ALL culture (specifically, ALL-5), and essentially identical results have been obtained with an independent c...

Cultured cells typically grow asynchronously and individual cells are present in different phases of the cell cycle. Some organisms exhibit naturally synchronous cell cycles or can be synchronized by specific physiological stimuli. For example, nuclei within giant plasmodia of the slime mold Physarum polycephalum divide in a highly synchronous fashion1, and cells of the green algae Desmodesmus quadricauda can be synchronized by alternating light and dark periods2. While such organisms offer unique experimental attributes, they inadequately model the complexities of mammalian cells. The ability to artificially synchronize mammalian cell populations has been central to advancing our understanding of cell cycle regulation and the molecular basis of cell cycle checkpoints. Common methods include serum deprivation, chemical block and release, or exploiting physical characteristics3,4. Withdrawal of serum often causes cells to enter quiescence, and the re-addition of serum promotes cell cycle reentry into the G1 phase5. Chemical inhibitors include agents such as excess thymidine or hydroxyurea which block cells at the G1/S boundary, or microtubule inhibitors which typically arrest cells in the M phase3,4. Approaches that exploit physical characteristics include mitotic shake-off which can be used to enrich for mitotic cells since they are less adherent than interphase cells6. However, all of these techniques have potential drawbacks. For example, not all cell types maintain viability in the absence of serum or the presence of chemical inhibitors, and the yield of cells after mitotic shake-off is limited without prior synchronization with mitotic inhibitors.
With the exception of early embryonic cell cycles, where cells progressively decrease in size as they divide7, most cells advancing through the cycle undergo growth phases and become larger. This property is exploited in the technique of counterflow centrifugal elutriation (CCE), which can be used to separate cells differing in size and hence in cell cycle phase8,9. During CCE, cells are under the influence of two opposing forces: centrifugal force, which drives the cells away from the axis of rotation, and fluid velocity (counterflow), which drives the cells towards the axis of rotation (Figure 1). Cells in the elutriation chamber reach an equilibrium position where these forces are equal. The key factors dictating the equilibrium position are cell diameter and density. As the flow rate of the buffer solution is increased and the counterflow drag force outweighs the centrifugal force, a new equilibrium is established, causing a change in position of cells inside the chamber. All cells are shifted toward the chamber exit, resulting in smaller ones leaving the chamber first, whereas the larger cells stay within the chamber until the counterflow rate is increased sufficiently to promote their exit. The cells escaping the elutriation chamber with consecutive increases in counterflow rate can be collected in specific fractions and each fraction contains cells of sequentially increasing sizes. Instead of conducting elutriation with incremental increases in counterflow rate, sequential decreases in centrifugal force by decreasing centrifugation speed will accomplish the same result. The process of elutriation requires optimization depending on cell type, elutriation buffer employed, and the specific apparatus used. The rate of sedimentation of cells under these conditions is best described by Stokes Law: SV = [d2(&#961;p-&#961;m)/18&#951;].&#969;2r, where SV = sedimentation velocity; d = diameter of the particle; &#961;p = density of the particle; &#961;m = density of the buffer; &#951; = viscosity of the buffer; &#969; = angular velocity of the rotor; and r = radial position of the particle. Thus, SV is proportional to cell diameter and density, and since diameter is raised to the second power, it contributes more significantly than density which is generally constant through the cell cycle.
An important advantage of CCE is that cells are not subjected to harsh conditions of chemical treatment or nutritional deprivation and can be recovered in excellent yield essentially unperturbed. A requirement is that the cells in question undergo a significant increase in diameter, of at least 30%, as they traverse the cell cycle. The main disadvantage is the cost of the specialized centrifuge, rotor, and accessories. Nonetheless, the ability to prepare cells enriched in specific cell cycle phases by CCE has greatly facilitated cell cycle research in organisms from yeast to mammalian cells9,10. Moreover, recent advances are expanding uses to the separation of cells from healthy versus cancerous tissue, the separation of different cell types in heterogeneous mixtures, and to the production of specific cell types for immunotherapy9.
Our major interest has been in understanding the mechanism of action of microtubule targeting agents (MTAs) such as vinca alkaloids and taxanes. These drugs were thought to act exclusively in mitosis, blocking spindle microtubule function11,12, but recent evidence suggests they may also target interphase microtubules13,14. We recently reported that primary acute lymphoblastic leukemia (ALL) cells undergo death in either the G1 phase or in the M phase when treated with vincristine and other MTAs in a cell cycle-dependent manner15. The ability to separate ALL cells into different cell cycle phases by CCE was instrumental in reaching this conclusion. In this article, we describe the technical details and offer practical tips for the application of CCE to the preparation of primary ALL cells in different cell cycle phases.

<table border="0" cellpadding="0" cellspacing="0" width="1542">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Hanks&#39; balanced salt solution</td>
			<td style="width:108px;">Lonza</td>
			<td style="width:119px;">10-508Q</td>
			<td style="width:780px;">1 L bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">Fetal Plus bovine serum</td>
			<td style="width:108px;">Atlas Biologicals</td>
			<td style="width:119px;">FP-0500-A</td>
			<td>500 mL bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">2-napthol-6,8-disulfonic acid dipotassium salt</td>
			<td style="width:108px;">Acros Organics</td>
			<td style="width:119px;">212-672-4</td>
			<td>100 g powder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">50 mL concial tubes</td>
			<td style="width:108px;">Denville Scientifics</td>
			<td style="width:119px;">C1062-P</td>
			<td>500/case</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">PES Membrane 0.22 &#181;m filter unit</td>
			<td style="width:108px;">Millipore</td>
			<td style="width:119px;">SLGP033RB</td>
			<td>250/pack</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Avanti J-26S XPI w/ Elut. Non-IVD - 50/60 Hz, 200/208/240V</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:119px;">B14544</td>
			<td>centrifuge compatible with elutriation system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">JE 5.0 elutriator rotor kit</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:119px;">356900</td>
			<td>rotor kit which includes the rotor, T-handle hex wrench, the quick release assembly (w/o the elutriation chamber), anchoring cable, and the strobe assembly</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Chamber, standard, 4-mL, &#34;A&#34;</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:119px;">356943</td>
			<td>standard elutriation chamber&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Masterflex L/S Easy-Load pump head</td>
			<td style="width:108px;">Cole-Parmer</td>
			<td style="width:119px;">EW-07518-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Masterflex L/S Variable-Speed Drive</td>
			<td style="width:108px;">Cole-Parmer</td>
			<td style="width:119px;">EW-07528-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Masterflex BioPharm platinum-cured silicone pump tubing, L/S 16</td>
			<td style="width:108px;">Cole-Parmer</td>
			<td style="width:119px;">EW-96420-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">25 G x 1.5 Precision Glide needle</td>
			<td style="width:108px;">BD</td>
			<td style="width:119px;">305127</td>
			<td>sterile, single-use needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">10 mL Luer-Lok syringe</td>
			<td style="width:108px;">BD</td>
			<td style="width:119px;">309604</td>
			<td>sterile, single-use syringes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">Vi-Cell XR</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:119px;">383556</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">PI/RNase staining buffer</td>
			<td style="width:108px;">BD Pharmingen</td>
			<td style="width:119px;">550825</td>
			<td>propidium iodide/RNase staining buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">cyclin B1 (GNS1) mouse monoclonal IgG antibody</td>
			<td style="width:108px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-245</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">cyclin D1 (DCS-6) mouse monoclonal IgG antibody</td>
			<td style="width:108px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-20044</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">P-Rb (S807/811) (D20B12) XPR rabbit monoclonal antibody</td>
			<td style="width:108px;">Cell Signaling Technology</td>
			<td style="width:119px;">8516s</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">GAPDH (14C10) rabbit monoclonal antibody</td>
			<td style="width:108px;">Cell Signaling Technology</td>
			<td style="width:119px;">2118s</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Goat anti-mouse IgG (H+L)-HRP conjugate</td>
			<td style="width:108px;">BioRad</td>
			<td style="width:119px;">170-6516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Goat anti-rabbit IgG (H+L)-HRP conjugate</td>
			<td style="width:108px;">BioRad</td>
			<td style="width:119px;">170-6515</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Primary ALL cells (3 - 4 x 108) were subjected to centrifugal elutriation and collected into two wash fractions and twenty main fractions, as described in the protocol. Table 1 shows representative data where the total number of cells in each fraction as well as the corresponding rotor speed are presented. Overall yield was typically over 80%. Measurement of cell diameter in individual fractions confirmed that cell diameter increased with advancing elutriation (<strong class="x...

Watch this Scientific Journal Video about Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation at JoVE.com

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

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 ...

The overall goal of this procedure, is to isolate cells into different cell cycle phases using centrifugal elutriation. This method can help answer key questions in the cancer therapeutics field. Because the isolation of cells into separate phases, allows for the interrogation of phase dependent affects of anti-cancer compounds.The main advantage of this technique is that it allows for separation of cells into specific phases using physical properties, rather than chemical perturbations. To start the experiment, install the strobe assembly into the centrifuge such that the power cord is fed out of the chamber through the port on the left. Adjust the strobe flash lamp so that it lines up with the viewing window, when the centrifuge is closed.Secure the assembly in place, by tightening two phillips head screws, at the top of each bracket. And then by tightening the thumb screws at the bottom of the bracket. Plug the power cord into the strobe power port, located at the back of the centrifuge.Next, set the rotors straight down onto the centrifuge drive hub, and firmly secure it with a T handle hex wrench. Then, place the assembly containing the elutriation chamber, counterbalance, rotating seal assembly, and a mounting plate onto the rotor. Push down evenly, placing one hand on the elutriation chamber, and the other hand on the counterbalance, until the bolts on the rotor snap into place.Place the pin, located at one end of the cable, into the hole in the rotating seal assembly. And secure the eyehole, at the other end, with one of the phillips head screws, affixed onto the bracket to connect the anchoring cable. To assemble the flow system, to pump buffer into and out of the elutriation chamber, connect size 16 tubing to a variable speed pump.Insert one free end of this tubing through a port at the top left of the centrifuge chamber, such that it enters into the chamber. Attach a fitting to the free end of this tube, and then insert the fitting into the input hole of the rotating seal assembly. Lastly, insert the output tubing, through a port, to attach it to the transfer tube at the top of the rotating seal assembly.Once the elutriation system is assembled, sterilize it by placing the other free end of the input tubing into a 500 milliliter bottle, filled with 5%bleach. And placing the output tubing into an empty one liter waste bottle. Next, turn on the pump, and allow the bleach to flow all the way through the elutriation system, until it is pumped out into the waste bottle.Now, turn the centrifuge on and allow it to come to the maximum designated speed to release bubbles and back pressure. Once the set speed is reached, stop the centrifuge. After the rotor has stopped completely, open the lid and inspect the chamber for any leaks.Next, in a similar fashion, as previously described, run sterile water through the system for 10 minutes. After flushing with water, replace the water reservoir with a sterile glass bottle filled with 100 milliliters of elutriation buffer. Place the input tubing into this bottle, so that it is at the very bottom.Start the centrifuge, and allow the buffer to flow through the system. After the reservoir bottle is close to empty, and water is fully flushed out of the system, move the output tubing from the waste bottle into the reservoir so that buffer is continuously recycled through the system. Finally, switch on the strobe lamp, and turn the elutriation knob to the extreme left.Now, adjust the viewing window by slowly turning the knob to the right, so that the elutriation chamber is clearly seen, and the strobe light is in sync with the rotor's speed. Prepare the cell sample as described in the text protocol. Add the resuspended cells into the reservoir bottle containing the recycling elutriation buffer.Allow the cells to enter into the system and reach equilibrium within the elutriation chamber. To ensure that all cells are retained in the chamber, take a drop of the eluent on a glass slide, and view it under a microscope. If the sample is devoid of intact cells, it confirms the retention in the chamber.Once all the cells have entered into the chamber, and a distinct boundary at the whitest part of the chamber, containing cells and equilibrium is seen, begin collecting fractions. Move the output tubing to a pre-chilled 50 milliliter conical tube, labeled W1.Collect 50 milliliters of elutriation buffer in this tube and store on ice. Soon after collecting the W1 fraction, lower the centrifuge speed to 821 G.And move the output tube to the conical tube, labeled W2, and collect 50 milliliters more at this speed.Continue changing the speed, and collecting fractions. Make sure there is medium in the reservoir tank, and always store the collected fractions on ice. After all fractions have been collected, stop the centrifuge, and collect 50 milliliters in a conical tube, labeled stop.Pellet cells collected in each of the fractions, and using downstream analysis, such as flow cytometry, or western blotting, as required. A line graph displaying the average diameter of cells in successive elutriation fractions, is shown. Note that the larger the cell diameter, the higher the fraction number at which it elutes.Results from fluorescents activated cells sorting analysis, show the DNA content of cells in each of the 20 elutriation fractions. Early fractions, F1 to F5, contain almost exclusively cells with 2N DNA content representing cells in G1 phase. A mixture of cells in G1 and S phases, was seen in intermediate fractions, F6 to F9.And cells with 4N DNA content, representing cells in G2 to M, were observed starting in F10.Shown here, are western blot results from probing the levels of cell cycle related proteins in pooled fractions of cells in G1 phase, and G2 to M phase. The G2 to M Pool had higher levels of Phospho-Rb and Cyclin B one, compared to the G1 Pool, as expected. Cyclin D one signal was weak, but nonetheless was detectable in the G1 Pool, and undetectable in the G2 to M Pool.Once mastered, this technique can be done in three hours if it is performed properly. While attempting this procedure, it is important to remember to take your time, and make sure that the elutriation system is assembled properly, and that all materials and solutions are prepared. Following this procedure, other methods such as cell cycle analysis, cell viability assays and immunoblots can be performed in order to interrogate how therapeutic compounds may be acting in a cell cycle phase dependent manner.

preparation-primary-acute-lymphoblastic-leukemia-cells-different-cell

Research

JoVE Journal

Cancer Research

8.1K visualizações.  University of Arkansas for Medical Sciences. The overall goal of this procedure, is to isolate cells into different cell cycle phases using centrifugal elutriation. This method can help answer key questions in the cancer therapeutics field. Because the isolation of cells into separate phases, allows for the interrogation of phase dependent affects of anti-cancer compounds.The main advantage of this technique is that it allows for separation of cells into specific phases using physical properties, rather than chemical perturbation...