Name: preparation of primary acute lymphoblastic leukemia cells in different cell cycle phases by centrifugal elutriation
Uploaded: 2017-11-10T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation at JoVE.com

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>
	<li>Beach, D., Piper, M., Shall, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+newly-initiated+DNA+from+the+early+S+phase+of+the+synchronous+eukaryote,+Physarum+polycephalum.">Isolation of newly-initiated DNA from the early S phase of the synchronous eukaryote, Physarum polycephalum.</a> Exp cell res. 129, 211-221 (1980).</li><li>Sevcikova, T., 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=Completion+of+cell+division+is+associated+with+maximum+telomerase+activity+in+naturally+synchronized+cultures+of+the+green+alga+Desmodesmus+quadricauda.">Completion of cell division is associated with maximum telomerase activity in naturally synchronized cultures of the green alga Desmodesmus quadricauda.</a> FEBS letters. 587, 743-748 (2013).</li><li>Rosner, M., Schipany, K., Hengstschlager, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merging+high-quality+biochemical+fractionation+with+a+refined+flow+cytometry+approach+to+monitor+nucleocytoplasmic+protein+expression+throughout+the+unperturbed+mammalian+cell+cycle.">Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle.</a> Nat protoc. 8, 602-626 (2013).</li><li>Banfalvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+cell+synchronization.">Overview of cell synchronization.</a> Meth mol biol. 761, 1-23 (2011).</li><li>Langan, T. J., Rodgers, K. R., Chou, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+Mammalian+Cell+Cultures+by+Serum+Deprivation.">Synchronization of Mammalian Cell Cultures by Serum Deprivation.</a> Meth mol biol. 1524, 97-105 (2017).</li><li>Dulla, K., Margalef, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-Scale+Mitotic+Cell+Synchronization.">Large-Scale Mitotic Cell Synchronization.</a> Meth mol biol. 1524, 65-74 (2017).</li><li>Siefert, J. C., Clowdus, E. A., Sansam, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+control+in+the+early+embryonic+development+of+aquatic+animal+species.">Cell cycle control in the early embryonic development of aquatic animal species.</a> Comp biochem physiol Toxicol,pharmacol CBP. 178, 8-15 (2015).</li><li>Banfalvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+synchronization+of+animal+cells+and+nuclei+by+centrifugal+elutriation.">Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation.</a> Nat protoc. 3, 663-673 (2008).</li><li>Grosse, J., Meier, K., Bauer, T. J., Eilles, C., Grimm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation+by+countercurrent+centrifugal+elutriation:+recent+developments.">Cell separation by countercurrent centrifugal elutriation: recent developments.</a> Prep biochem,biotechnol. 42, 217-233 (2012).</li><li>Smith, J., Manukyan, A., Hua, H., Dungrawala, H., Schneider, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+Yeast.">Synchronization of Yeast.</a> Meth mol. 1524, 215-242 (2017).</li><li>Gascoigne, K. E., Taylor, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+anti-mitotic+drugs+kill+cancer+cells?.">How do anti-mitotic drugs kill cancer cells?.</a> J cell sci. 122, 2579-2585 (2009).</li><li>Manchado, E., Guillamot, M., Malumbres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+cells+by+targeting+mitosis.">Killing cells by targeting mitosis.</a> Cell death diff. 19, 369-377 (2012).</li><li>Komlodi-Pasztor, E., Sackett, D., Wilkerson, J., Fojo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis+is+not+a+key+target+of+microtubule+agents+in+patient+tumors.">Mitosis is not a key target of microtubule agents in patient tumors.</a> Nature reviews. Clinic oncol. 8, 244-250 (2011).</li><li>Furst, R., Vollmar, 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=A+new+perspective+on+old+drugs:+non-mitotic+actions+of+tubulin-binding+drugs+play+a+major+role+in+cancer+treatment.">A new perspective on old drugs: non-mitotic actions of tubulin-binding drugs play a major role in cancer treatment.</a> Die Pharmazie. 68, 478-483 (2013).</li><li>Kothari, A., Hittelman, W. N., Chambers, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Cycle-Dependent+Mechanisms+Underlie+Vincristine-Induced+Death+of+Primary+Acute+Lymphoblastic+Leukemia+Cells.">Cell Cycle-Dependent Mechanisms Underlie Vincristine-Induced Death of Primary Acute Lymphoblastic Leukemia Cells.</a> Cancer res. 76, 3553-3561 (2016).</li><li>Nijmeijer, B. A., 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=Long-term+culture+of+primary+human+lymphoblastic+leukemia+cells+in+the+absence+of+serum+or+hematopoietic+growth+factors.">Long-term culture of primary human lymphoblastic leukemia cells in the absence of serum or hematopoietic growth factors.</a> Exp hematol. 37, 376-385 (2009).</li><li>Knudsen, E. S., Wang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+mechanisms+for+the+inhibition+of+E2F+binding+to+RB+by+cyclin-dependent+kinase-mediated+RB+phosphorylation.">Dual mechanisms for the inhibition of E2F binding to RB by cyclin-dependent kinase-mediated RB phosphorylation.</a> Mol Cell Bio. 17, 5771-5783 (1997).</li><li>Malumbres, M., Barbacid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cyclin-dependent+kinases.">Mammalian cyclin-dependent kinases.</a> Trends biochem sci. 30, 630-641 (2005).</li><li>Liang, H., Hittelman, W., Nagarajan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitous+expression+and+cell+cycle+regulation+of+the+protein+kinase+PIM-1.">Ubiquitous expression and cell cycle regulation of the protein kinase PIM-1.</a> Arch biochem biophys. 330, 259-265 (1996).</li><li>Sen, 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=Expression+of+a+gene+encoding+a+tRNA+synthetase-like+protein+is+enhanced+in+tumorigenic+human+myeloid+leukemia+cells+and+is+cell+cycle+stage-+and+differentiation-dependent.">Expression of a gene encoding a tRNA synthetase-like protein is enhanced in tumorigenic human myeloid leukemia cells and is cell cycle stage- and differentiation-dependent.</a> Proc Natl Acad Sci USA. 94, 6164-6169 (1997).</li><li>Ly, T., Endo, A., Lamond, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+the+response+to+cell+cycle+arrests+in+human+myeloid+leukemia+cells.">Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells.</a> eLife. 4, (2015).</li></ol>

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

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

Cell Cycle-specific Measurement of &amp;#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

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 ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...