Name: hplc-based assay to monitor extracellular nucleotide/nucleoside metabolism in human chronic lymphocytic leukemia cells
Uploaded: 2016-07-20T13:00:00
Description: Watch this Scientific Journal Video about HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells at JoVE.com

This work is supported by Associazione Italiana Ricerca Cancro (IG #12754).

CLL blood samples are obtained in accordance with Institutional Guidelines and Declaration of Helsinki.
1. Isolation of Leukemic Lymphocytes from Blood Samples of CLL Patients&#160;
<ol>
	<li>Collect blood sample in sodium heparin (green-top) tube17.</li>
	<li>Make 1: 3 dilution of whole blood with RT 1x phosphate buffered saline (PBS).</li>
	<li>Purify peripheral blood mononuclear cells (PBMC) from blood samples by density gradient centrifugation.
	<ol>
		<li>Underlay 5 ml of density centrifugation m...

<ol>
	<li>Naganuma, M., Wiznerowicz, E. B., Lappas, C. M., Linden, J., Worthington, M. T., Ernst, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+Critical+role+for+A2A+adenosine+receptors+in+the+T+cell-mediated+regulation+of+colitis.">Cutting edge: Critical role for A2A adenosine receptors in the T cell-mediated regulation of colitis.</a> J Immunology. 177 (5), 2765-2769 (2006).</li><li>Nemeth, Z. H., 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=Adenosine+receptor+activation+ameliorates+type+1+diabetes.">Adenosine receptor activation ameliorates type 1 diabetes.</a> FASEB J. 21 (10), 2379-2388 (2007).</li><li>Fan, M., Jamal Mustafa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+adenosine+in+airway+inflammation+in+an+allergic+mouse+model+of+asthma.">Role of adenosine in airway inflammation in an allergic mouse model of asthma.</a> Int Immunopharmacol. 6 (1), 36-45 (2006).</li><li>Csoka, B., 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=A2B+adenosine+receptors+protect+against+sepsis-induced+mortality+by+dampening+excessive+inflammation.">A2B adenosine receptors protect against sepsis-induced mortality by dampening excessive inflammation.</a> J Immunol. 185 (1), 542-550 (2010).</li><li>Peart, J. N., Headrick, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosinergic+cardioprotection:+multiple+receptors,+multiple+pathways.">Adenosinergic cardioprotection: multiple receptors, multiple pathways.</a> Pharmacol Ther. 114 (2), 208-221 (2007).</li><li>Ohta, 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=A2A+adenosine+receptor+protects+tumors+from+antitumor+T+cells.">A2A adenosine receptor protects tumors from antitumor T cells.</a> Proc Natl Acad Sci U S A. 103 (35), 13132-13137 (2006).</li><li>Hasko, G., Linden, J., Cronstein, B., Pacher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+receptors:+therapeutic+aspects+for+inflammatory+and+immune+diseases.">Adenosine receptors: therapeutic aspects for inflammatory and immune diseases.</a> Nat Rev Drug Discov. 7 (9), 759-770 (2008).</li><li>Cronstein, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine,+an+endogenous+anti-inflammatory+agent.">Adenosine, an endogenous anti-inflammatory agent.</a> J Appl Physiol (1985). 76 (1), 5-13 (1994).</li><li>Molina-Arcas, M., Casado, F. J., Pastor-Anglada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleoside+transporter+proteins.">Nucleoside transporter proteins.</a> Curr Vasc Pharmacol. 7 (4), 426-434 (2009).</li><li>Deaglio, 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=Adenosine+generation+catalyzed+by+CD39+and+CD73+expressed+on+regulatory+T+cells+mediates+immune+suppression.">Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression.</a> J Exp Med. 204 (6), 1257-1265 (2007).</li><li>Linden, J. <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+leukocyte+function+by+adenosine+receptors.">Regulation of leukocyte function by adenosine receptors.</a> Adv Pharmacol. 61, 95-114 (2011).</li><li>Antonioli, L., Blandizzi, C., Pacher, P., Hasko, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity,+inflammation+and+cancer:+a+leading+role+for+adenosine.">Immunity, inflammation and cancer: a leading role for adenosine.</a> Nat Rev Cancer. 13 (12), 842-857 (2013).</li><li>Antonioli, L., Csoka, B., Fornai, M., 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=Adenosine+and+inflammation:+what's+new+on+the+horizon.">Adenosine and inflammation: what's new on the horizon.</a> Drug Discov Today. 19 (8), 1051-1068 (1051).</li><li>Chiorazzi, N., Rai, K. R., Ferrarini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+lymphocytic+leukemia.">Chronic lymphocytic leukemia.</a> N Engl J Med. 352 (8), 804-815 (2005).</li><li>Abousamra, N. K., Salah El-Din, M., Hamza Elzahaf, E., Esmael, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectonucleoside+triphosphate+diphosphohydrolase-1+(E-NTPDase1/CD39)+as+a+new+prognostic+marker+in+chronic+lymphocytic+leukemia.">Ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1/CD39) as a new prognostic marker in chronic lymphocytic leukemia.</a> Leuk Lymphoma. 56 (1), 113-119 (2015).</li><li>Serra, 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=CD73-generated+extracellular+adenosine+in+chronic+lymphocytic+leukemia+creates+local+conditions+counteracting+drug-induced+cell+death.">CD73-generated extracellular adenosine in chronic lymphocytic leukemia creates local conditions counteracting drug-induced cell death.</a> Blood. 118 (23), 6141-6152 (2011).</li><li>Chen, L. S., Keating, M. J., Gandhi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+collection+methods+affect+cellular+protein+integrity:+implications+for+clinical+trial+biomarkers+and+ZAP-70+inn+CLL.">Blood collection methods affect cellular protein integrity: implications for clinical trial biomarkers and ZAP-70 inn CLL.</a> Blood. 124 (7), 1192-1195 (2014).</li><li>Kalina, 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=EuroFlow+standardization+of+flow+cytometer+instrument+settings+and+immunophenotyping+protocols.">EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols.</a> Leukemia. 26 (9), 1986-2010 (2012).</li><li>Deaglio, 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=CD38+and+ZAP-70+are+functionally+linked+and+mark+CLL+cells+with+high+migratory+potential.">CD38 and ZAP-70 are functionally linked and mark CLL cells with high migratory potential.</a> Blood. 110 (12), 4012-4021 (2007).</li><li>Sachsenmeier, K. F., 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=Development+of+a+novel+ectonucleotidase+assay+suitable+for+high-throughput+screening.">Development of a novel ectonucleotidase assay suitable for high-throughput screening.</a> J Biomol Screen. 17 (7), 993-998 (2012).</li></ol>

The protocol described here permits to evaluate the activity of the CD39/CD73 adenosinergic machinery in cell culture media from purified human leukemic cells. Through this HPLC method we can follow and quantitatively measure the enzymatic generation of ADO (CD73-dependent) and its subsequent degradation to INO (CD26/ADA dependent). The use of enzyme inhibitors allows to control the protocol and to have internal controls. The advantages and novelties of this protocol are that i) it may be applied to cells that are growin...

Adenosine (ADO) is a purine nucleoside with an adenine molecule attached to a ribose sugar molecule moiety through a glycosidic bond. When present in the extracellular environment, it protects cells from excessive damage by the action of the immune system. This role has been highlighted using different disease models, such as colitis1, diabetes2, asthma3, sepsis4, and ischemic injury5. One of the main ADO functions is the inhibition of immune responses in the tumor microenvironment, contributing to tumor immune evasion6. For this reason, the mechanisms involved in ADO formation and signaling are of considerable therapeutic interest7.
ADO levels in the tissue microenvironment are relatively low under normal physiologic conditions and certainly below the sensitivity threshold of immune cells. However, during hypoxia, ischemia, inflammation, infection, metabolic stress and tumor transformation they rapidly increase8. The elevated extracellular ADO levels in response to tissue-perturbing signals have a dual function: to report tissue injury in an autocrine and paracrine way and to generate tissue responses that can be generally viewed as cytoprotective.
Extracellular ADO can be formed through a variety of mechanisms, which include release from intracellular compartments mediated by nucleoside transporters9 or accumulation because of impaired degradation operated by adenosine deaminase. The main pathway leading to increased extracellular ADO levels involves the action of a cascade of ectonucleotidases, which are membrane associated ectoenzymes generating ADO by phosphohydrolysis of nucleotides released from dead or dying cells. This pathway proceeds through the sequential action of CD39 (ectonucleoside triphosphate diphosphohydrolase-1) that converts extracellular adenosine 5&#39;-triphosphate (ATP) or adenosine 5&#39;-diphosphate (ADP) to adenosine 5&#39;-monophosphate (AMP) and of CD73 (5&#39;-nucleotidase), which converts AMP to ADO10.
Extracellular ADO elicits its physiological responses by binding to four transmembrane ADO receptors, namely A1, A2A, A2B and A3. Each receptor has different affinities for ADO and specific tissue distribution. All the receptors have seven transmembrane domains and are G-protein coupled to intracellular GTP-binding proteins (G proteins), that can induce (Gs protein) or inhibit (Gi protein) adenylate cyclase activity and, subsequently, the production of intracellular cAMP. Therefore, changes in cytoplasmic cAMP levels impact on intracellular protein kinase activity during physiological responses11. Under physiological conditions extracellular ADO is below 1 &#181;M, which can activate indiscriminately A1, A2A and A3 receptors. However, the activation of A2B subtype requires considerably higher concentrations of the nucleoside, such as those generated under pathophysiological conditions. Alternatively, extracellular ADO can be degraded to inosine (INO) by adenosine deaminase (ADA) and CD26, an ADA complexing protein localizing ADA on the cell surface. Another possibility is that ADO is internalized by the cell through the equilibrative nucleoside transporters (ENT) and phosphorylated to AMP by ADO kinase protein12,13.
The aim of this protocol is to describe an analytical method of reverse phase high-performance liquid chromatography (RP-HPLC) to quantify in a single run the substrate AMP and the products ADO and INO, as generated by human lymphocytes. Our experience was initially obtained using cells from chronic lymphocytic leukemia (CLL) patients, which are characterized by the expansion of a mature population of CD19+/CD5+ B lymphocytes constitutively expressing CD3914,15. We showed approximately 30% of CLL patients express the CD73 ectoenzyme and that this phenotype correlates with a poor prognosis16. This subpopulation of leukemic cells co-expressing CD39 and CD73 can actively produce extracellular ADO from ADP and/or AMP. Preincubation of CD73+ CLL cells with &#945;,&#946;-methylene-ADP (APCP), a known inhibitor of CD73 enzymatic activity, completely blocks extracellular ADO synthesis confirming that CD73 represents the bottle-neck enzyme of that cascade16.
CLL cells also express ADA and the ADA complexing protein CD26, which are responsible for the conversion of ADO into INO. By using specific ADA inhibitors, such as erythro-9-(2-Hydroxy-3-nonyl)I wiadenine (EHNA) hydrochloride and deoxycoformycin (dCF), it is possible to block extracellular ADO degradation into INO. Furthermore, pretreatment with an ADA inhibitor in combination with dipyridamole, that blocks nucleoside transporters, enhances ADO accumulation in cell supernatants.
We have then extended this protocol to cells derived from other lineages, including T lymphocytes and myeloid cells, confirming CD73-dependent ADO production. These findings suggest that this HPLC protocol is highly versatile and that it can be applied to different cell lineages and to different culture conditions (Figure 1).
<img alt="Figure 1" src="/files/ftp_upload/54124/54124fig1.jpg" /> 
Figure 1.&#160;Schematic representation of the enzymatic machinery responsible for extracellular ADO production. Adenosine 5&#39;-triphosphate (ATP) and/or adenosine 5&#39;-diphosphate (ADP) can be degraded by CD39 to adenosine 5&#39;-monophosphate (AMP), which in turn is converted by CD73 into the nucleoside adenosine (ADO). Once ADO is produced in the extracellular space, it may reenter the cell through the nucleoside transporters (ENT), be converted into inosine (INO) or bind to different types of P1 ADO receptors. <a href="https://www.jove.com/files/ftp_upload/54124/54124fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Human blood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q water</td>
			<td>Millipore</td>
			<td></td>
			<td>double deionised water</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE-Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-CD3, -CD14, -CD16</td>
			<td>made in-house</td>
			<td></td>
			<td>mouse monoclonal</td>
		</tr>
		<tr>
			<td>PE-labeled anti-CD19</td>
			<td>Miltenyi Biotec</td>
			<td>120-014-229</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-labeled anti-CD5</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-574</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads sheep anti-mouse IgG</td>
			<td>Invitrogen</td>
			<td>11031</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Amresco</td>
			<td>E404-200TABS</td>
			<td>tablets</td>
		</tr>
		<tr>
			<td>bovine serum albumin (BSA)</td>
			<td>ID bio</td>
			<td>1000-70</td>
			<td>standard grade</td>
		</tr>
		<tr>
			<td>isolation buffer</td>
			<td></td>
			<td></td>
			<td>PBS 0.1 % BSA 2 mM EDTA, pH 7.4</td>
		</tr>
		<tr>
			<td>AIM V serum free medium</td>
			<td>GIBCO</td>
			<td>12055-091</td>
			<td>liquid (research grade)</td>
		</tr>
		<tr>
			<td>adenosine 5&#8217;-diphosphate (ADP)</td>
			<td>Sigma-Aldrich</td>
			<td>A2754</td>
			<td></td>
		</tr>
		<tr>
			<td>adenosine 5&#8217;-monosphate (AMP)</td>
			<td>Sigma-Aldrich</td>
			<td>A1752</td>
			<td></td>
		</tr>
		<tr>
			<td>adenosine (ADO)</td>
			<td>Sigma-Aldrich</td>
			<td>A9251</td>
			<td></td>
		</tr>
		<tr>
			<td>inosine (INO)</td>
			<td>Sigma-Aldrich</td>
			<td>I4125</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;,&#946;-methylene-ADP (APCP)</td>
			<td>Sigma-Aldrich</td>
			<td>M8386</td>
			<td>CD73 inhibitor</td>
		</tr>
		<tr>
			<td>EHNA hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>E114</td>
			<td>adenosine deaminase inhibitor</td>
		</tr>
		<tr>
			<td>Deoxycoformycin (dCF)</td>
			<td>Tocris</td>
			<td>2033</td>
			<td>adenosine deaminase inhibitor</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Dipyridamole</td>
			<td>Sigma-Aldrich</td>
			<td>D9766</td>
			<td>nucleoside transporter inhibitor</td>
		</tr>
		<tr>
			<td>acetonitrile (CHROMASOLV Plus)</td>
			<td>Sigma-Aldrich</td>
			<td>34998</td>
			<td>HPLC-grade</td>
		</tr>
		<tr>
			<td>ammonium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>9688</td>
			<td>7 mM, pH 3.0</td>
		</tr>
		<tr>
			<td>hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>30721-1L</td>
			<td>min. 37 %</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Name</td>
			<td style="width:168px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:235px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Equipment</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B&#252;rker cell counter</td>
			<td>VWR</td>
			<td style="width:119px;">631-0920</td>
			<td>hemocytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-15 Magnet</td>
			<td>Invitrogen</td>
			<td style="width:119px;">12301D</td>
			<td>Dynal magnetic bead separator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microcentrifuge safe-lock tubes</td>
			<td>Eppendorf</td>
			<td>030-120-0086</td>
			<td>1.5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PET centrifuge tubes</td>
			<td>Corning</td>
			<td>430053/430304</td>
			<td>15 &#8211; 50 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minisart RC4 syringe filters</td>
			<td>Sartorius Stedim Biotech</td>
			<td>17821</td>
			<td>membrane 0.2 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">short thread vials</td>
			<td>VWR</td>
			<td>548-0029</td>
			<td>1.5 ml/glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro-inserts</td>
			<td>VWR</td>
			<td>548-0006</td>
			<td>0.1 ml/glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">screw caps</td>
			<td>VWR</td>
			<td>548-0085</td>
			<td>9 mm/PP blue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Atlantis dC18 Column</td>
			<td style="width:168px;">Waters</td>
			<td style="width:119px;">186001344</td>
			<td>5 &#181;m, 4.6 x 150 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Atlantis dC18 Guard Column</td>
			<td style="width:168px;">Waters</td>
			<td style="width:119px;">186001323</td>
			<td>5 &#181;m, 4.6 x 20 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waters Alliance 2965 Separations Module</td>
			<td style="width:168px;">Waters</td>
			<td style="width:119px;"></td>
			<td>HPLC separation module</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waters 2998 Photodiode Array (PDA) Detector</td>
			<td style="width:168px;">Waters</td>
			<td style="width:119px;"></td>
			<td>UV detector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Waters Empower2 software</td>
			<td style="width:168px;">Waters</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

hplc-based assay to monitor extracellular nucleotide/nucleoside metabolism in human chronic lymphocytic leukemia cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

To evaluate the percentage (%) of leukemic cells in freshly purified PBMCs from a representative CLL patient, cells are marked with anti-CD19 and anti-CD5 antibodies. The left panel of Figure 3 represents a cytofluorimetric dot plot with a selective gate on live cells. Figure 3 shows an example of PBMC from a CLL patient before (middle panel) and after (right panel) B cell purification.
An examp...

Watch this Scientific Journal Video about HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells at JoVE.com

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

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Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of FÃƒÆ’Ã‚Â¢Ãƒâ€¹Ã¢â‚¬Â '

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests ...

Mass Spectrometry-Guided Genome Mining as a Tool to Uncover Novel Natural Products

The chemical space covered by natural products is immense and widely unrecognized. Therefore, convenient methodologies to perform wide-ranging evaluation ...