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 media (e.g., Ficoll) in a 15 ml centrifuge tube and carefully transfer 10 ml of diluted blood. Immediately centrifuge at 1,500 x g for 20 min at RT.</li>
	</ol>
	</li>
	<li>Aspirate part of the supernatant with a glass Pasteur pipette connected to a vacuum pump and collect the PBMC ring at the interphase between density centrifugation media and diluted plasma with a 5 ml pipette. Transfer to a 50 ml centrifuge tube and wash with PBS (50 ml final volume). Centrifuge at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant with a glass Pasteur pipette, resuspend the pellet in 50 ml of PBS and count the cells using a hemocytometer.</li>
	<li>Stain PBMCs with anti-CD19 and -CD5 antibodies for flow cytometry following a standard protocol for immunostaining and check for PBMC subpopulation composition18. 
	Note: CLL lymphocytes are CD19+/CD5+.</li>
	<li>Check the % of CD19+/CD5+ cells using a flow cytometer18. Purify B lymphocytes from patients with a &#8805;95% of CD19+/CD5+ leukemic cells following the instructions detailed in step 2 for the negative isolation of B cells19.</li>
</ol>
2. Purification of Leukemic B Cells by Negative Isolation
<ol>
	<li>Resuspend 107 PBMCs/ml in PBS 0.1 % bovine serum albumin (BSA) 2 mM EDTA, pH 7.4 (isolation buffer).</li>
	<li>Add 10 &#181;g of mouse monoclonal anti-CD3, -CD14 and -CD16 primary antibodies for 107 cells and incubate for 30 min at 4 &#176;C.</li>
	<li>Wash the cells with the isolation buffer and centrifuge at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the cells in the isolation buffer at 107 PBMCs/ml.</li>
	<li>Before incubating with the cells, resuspend sheep anti-mouse IgG magnetic beads in the vial. Transfer 100 &#181;l of beads per 107 cells to a tube.</li>
	<li>Add 1 mL of isolation buffer and place the tube in a magnet holder for 1 min. Discard the supernatant with a 5 ml pipette.</li>
	<li>Remove the tube from the magnet and resuspend the washed beads in 100 &#181;l of isolation buffer.</li>
	<li>Incubate 107 cells with 100 &#181;l of magnetic beads for 30 min at 4 &#176;C with gentle tilting and rotation.</li>
	<li>Place the tube in the magnet holder for 2 min and transfer the supernatant with the CD19+/CD5+ unbound cells to a fresh tube with a 5 ml pipette.</li>
	<li>At the end of the isolation protocol, stain 100 &#181;l of cells with anti-CD19 and -CD5 antibodies for flow cytometry and incubate 30 min at 4 &#176;C. Wash the cells with 3 ml of PBS 1% BSA, discard the excess of supernatant and check B cell purity again by flow cytometry19. 
	Note: Perform an additional negative isolation step if B cell purity is lower than 95%.</li>
</ol>
3. Preparation of Standard and Inhibitors Stock Solutions
<ol>
	<li>To prepare AMP, weigh out 0.00345 g of AMP and dissolve it in 5 ml of serum-free medium to have a 2 mM standard solution of AMP.</li>
	<li>To prepare ADO, weigh out 0.00265 g of ADO and dissolve it in 5 ml of serum-free medium to have a 2 mM standard solution of ADO.</li>
	<li>To prepare INO, weigh out 0.00268 g of INO and dissolve it in 5 ml of serum-free medium to have a 2 mM standard solution of INO.</li>
	<li>Prepare a 400 &#181;M solution of AMP, ADO and INO by mixing 1 ml of the 2 mM stock solution in 4 ml of serum-free medium (5 ml final volume).
	<ol>
		<li>Make 1:4 dilution of each 400 &#181;M standard solution to obtain a 100 &#181;M concentration by pipetting 500 &#181;l of the 400 &#181;M solution in 1,500 &#181;l of serum-free medium (2 ml final volume).</li>
		<li>Prepare serial dilutions of each compound in serum-free medium to obtain the following concentrations: 100 &#181;M - 50 &#181;M - 25 &#181;M - 10 &#181;M - 5 &#181;M - 2.5 &#181;M - 1 &#181;M - 0.5 &#181;M - 0.25 &#181;M. 
		Note: For example, pipette 1 ml of the 100 &#181;M solution in 1 ml of serum-free medium (1:2 dilution) to obtain 50 &#181;M concentration and proceed with the remaining dilutions.</li>
	</ol>
	</li>
	<li>Prepare a 10 mM stock solution of APCP dissolved in PBS; aliquot and stock at - 30 &#176;C.</li>
	<li>Prepare 10 mM stock solutions of EHNA hydrochloride, dCF and dipyridamole dissolved in dimethyl sulfoxide (DMSO); aliquot and stock at -30 &#176;C.</li>
</ol>
4. Program the HPLC Method
<ol>
	<li>Prepare a 7 mM ammonium acetate buffer by dissolving 0.770 g of ammonium acetate in 2 L of double deionised water (Buffer A). Adjust to pH 3.0 with hydrochloric acid.</li>
	<li>Prepare a glass bottle containing at least 2 L of ultrapure acetonitrile for HPLC (Buffer B) and connect the guard column and the column to the HPLC. 
	Note: The columns and buffers are at RT while in use.</li>
	<li>Log into the HPLC software and select the &#34;browse project&#34; button. Go to the &#34;file&#34; menu and select &#34;new method&#34; and then &#34;instrument method&#34;.</li>
	<li>Program the equilibration column method as per Table 1. Set a flow rate of 1.00 ml/min and program the UV detector to read at 260 nm. Save the instrument method and select again &#8220;new method&#8221; from the &#8220;file&#8221; menu and then &#8220;method set&#8221;. Choose the instrument method previously saved and save the current method set with the same name.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time</td>
			<td>Flow rate (ml/min)</td>
			<td>%A</td>
			<td>%B</td>
		</tr>
		<tr>
			<td></td>
			<td>1.00</td>
			<td>100</td>
			<td>0</td>
		</tr>
		<tr>
			<td>1.24</td>
			<td>1.00</td>
			<td>100</td>
			<td>0</td>
		</tr>
		<tr>
			<td>6.22</td>
			<td>1.00</td>
			<td>2</td>
			<td>98</td>
		</tr>
		<tr>
			<td>18.65</td>
			<td>1.00</td>
			<td>2</td>
			<td>98</td>
		</tr>
	</tbody>
</table>
Table 1: Equilibration column method. Schematic representation of solvent changes for the equilibration of the column. Buffer A: 7 mM ammonium acetate, pH 3.0. Buffer B: acetonitrile.
<ol start="5">
	<li>Repeat the step 4.3 to program the run sample method indicated in Table 2. Set a flow rate of 1.00 mL/min and program the UV detector to read at 260 nm. Save the instrument method and repeat the same procedure as described in step 4.4 to save the method set..</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time</td>
			<td>Flow rate (ml/min)</td>
			<td>%A</td>
			<td>%B</td>
		</tr>
		<tr>
			<td></td>
			<td>1.00</td>
			<td>0</td>
			<td>100</td>
		</tr>
		<tr>
			<td>3.74</td>
			<td>1.00</td>
			<td>0</td>
			<td>100</td>
		</tr>
		<tr>
			<td>13.71</td>
			<td>1.00</td>
			<td>15</td>
			<td>85</td>
		</tr>
		<tr>
			<td>17.00</td>
			<td>1.00</td>
			<td>100</td>
			<td>0</td>
		</tr>
		<tr>
			<td>20.00</td>
			<td>1.00</td>
			<td>100</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 2: Run sample method. Schematic representation of solvent changes for HPLC measurement of purine compounds. Buffer A: 7 mM ammonium acetate, pH 3.0. Buffer B: acetonitrile.&#160;
<ol start="6">
	<li>Select the &#34;run samples&#34; button, choose &#34;new sample set method&#34; from the &#34;file&#34; menu.</li>
	<li>Select the &#34;empty&#34; option and enter the number of the vial (in the autosampler), the sample name, the injection volume (50 &#181;l). Select the method set saved before for the &#34;method set&#34; column and enter the total run time (min). 
	Note: Enter the equilibration column method (Table 1) in the &#34;method set&#34; column before each sample run and enter the total run time (min).</li>
</ol>
5. Generation of a Standard Calibration Curve for Each Compound
<ol>
	<li>Inject 50 &#181;l of blank (specific serum-free medium, which does not contain adenosine deaminase) and of each standard sample following the methods described in Table 1 and Table 2.
	<ol>
		<li>Use the equilibration column method (Table 1) prior to each sample run and set the gradient conditions described in Table 2 to obtain adequate peak separation. Refer to Table 3 for reported retention times of AMP, ADO and INO under these HPLC conditions.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Retention Time</td>
			<td>&#955;max</td>
		</tr>
		<tr>
			<td>AMP</td>
			<td>8.00 min</td>
			<td>260</td>
		</tr>
		<tr>
			<td>INO</td>
			<td>11.00 min</td>
			<td>260</td>
		</tr>
		<tr>
			<td>ADO</td>
			<td>11.90 min</td>
			<td>260</td>
		</tr>
	</tbody>
</table>
Table 3: Retention times of purine compounds. Typical retention times observed for AMP, ADO and INO. The UV detector is programmed to read at 260 nm.&#160;
<ol start="2">
	<li>Determine the peak area of AMP, ADO and INO at different concentrations choosing the &#34;process&#34; button in the HPLC software and next the &#34;integrate&#34; option. Alternatively select manually the starting and end points of each peak.</li>
	<li>Plot the peak areas against the nominal concentration of each standard to obtain the nine-point calibration curve (100, 50, 25, 10, 5, 2.5, 1, 0.5, 0.25 &#181;M).</li>
	<li>Calculate the equation of a straight line for AMP, ADO and INO: y = mx + b, 
	where x is the &#181;M concentration and y correspond to the peak area.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54124/54124fig2.jpg" /> 
Figure 2.&#160;Generation of an internal standard curve. Representative calibration standard curve for ADO and the relative equation obtained. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54124/54124fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Pretreatment with the Inhibitors and Incubation with the Substrate (AMP)
<ol>
	<li>To test AMP consumption, ADO and INO generation without inhibition of CD73, ADA and nucleoside transporters, resuspend 2 x 106 CD19+/CD5+ CLL cells (isolated and purified in steps 1 and 2) in 250 &#181;l of serum-free medium and plate cells in a 48 well plate (or in a 1.5 ml microcentrifuge tube).</li>
	<li>To block CD73 enzymatic activity, dilute 1:1,000 the stock solution of APCP (10 mM) in the culture medium to obtain a 10 &#181;M final concentration. Resuspend 2 x 106 CLL cells in 250 &#181;l of culture medium containing APCP and pretreat 60 min at 37 &#176;C in a cell culture incubator.</li>
	<li>To block the conversion of ADO into INO, dilute 1:1,000 the stock solution of EHNA hydrochloride and/or dCF (both 10 mM) in serum-free medium to have a 10 &#181;M final concentration. Resuspend 2 x 106 CLL cells in 250 &#181;l of culture medium containing EHNA and/or dCF. Incubate for 30 min at 37 &#176;C in the cell culture incubator.</li>
	<li>To block the uptake of ADO through the nucleoside transporters, dilute 1:1,000 the stock solution of dipyridamole (10 mM) in serum-free medium to have a 10 &#181;M final concentration. Resuspend 2 x 106 CLL cells in 250 &#181;l of culture medium containing dipyridamole and incubate for 30 min at 37 &#176;C in the cell culture incubator.</li>
	<li>Prepare single solutions of APCP, EHNA, dCF and dipyridamole diluted 1:1,000 in 400 &#181;M AMP.</li>
	<li>Add 250 &#181;l of 400 &#181;M AMP or AMP plus inhibitors to obtain a final concentration of 200 &#181;M AMP. Include a condition in the absence of the substrate to be used as blank sample.</li>
	<li>Incubate from 30 to 60 min at 37 &#176;C. 
	Note: The optimal concentration of AMP and the incubation time were experimentally determined.</li>
	<li>At the end of the incubation time, collect 500 &#181;l of the supernatants in cold microcentrifuge tubes (on ice) and immediately centrifuge at 17,000 x g at 4 &#176;C for 5 min.</li>
	<li>Transfer the supernatants in new 1.5 ml microcentrifuge tubes and immediately store at -80 &#176;C or proceed to sample preparation for the HPLC runs.</li>
</ol>
7. Samples Preparation for HPLC
<ol>
	<li>Filter the supernatants in new 1.5 ml microcentrifuge tubes with the 0.2 &#181;m syringe filters. Use 1 ml syringes for filtering.</li>
	<li>If the HPLC system is provided with an autosampler, prepare the glass vials for HPLC and use the 0.1 ml micro-inserts for small volumes of sample.</li>
	<li>Transfer at least 100 &#181;l of sample in the glass vial with a micropipette or a glass Pasteur pipette. Be careful to transfer without bubbles and close the vial with the screw cap.</li>
	<li>Samples are now ready for analysis by RP-HPLC.</li>
</ol>
8. HPLC Measurements of Purines&#160;
<ol>
	<li>Select the &#34;run samples&#34; button; choose &#34;new sample set method&#34; from the &#34;file&#34; menu.</li>
	<li>Select the &#34;empty&#34; option and indicate: the number of the vial (in the autosampler), the sample name, the injection volume (50 &#181;l).</li>
	<li>Select the equilibration column method and the run sample method described in Table 1 and Table 2, respectively, for all the blank and sample runs that follow. 
	Note: Set the equilibration column method (Table 1) before each sample run.</li>
	<li>Inject 50 &#181;l of blank (serum-free medium) and of each sample.</li>
	<li>Determine the concentration of purines in each sample, quantify the AMP, ADO and INO by obtaining the peak areas at the retention times described in Table 3.
	<ol>
		<li>Select the &#34;process&#34; button and next the &#34;integrate&#34; option. Alternatively choose manually the start and end points of each single peak to obtain the area measurement. This is done by tracing a line between the start and end points of the peak.</li>
		<li>Determine the &#181;M concentration applying the equation obtained for the standard curve: y = mx + b, where x represents the &#181;M concentration and y is the area of the peak measured from the unknown sample. 
		Note: An example of the ADO calibration standard curve is reported in Figure 2, where: x = (y area - 981.02)/40026</li>
	</ol>
	</li>
	<li>Considering that 2 x 106 cells were resuspended in 0.500 ml of medium, calculate&#160;the &#181;moles of AMP, ADO and INO consumed and/or produced by 106 CLL cells applying the following proportion: 
	&#181;moles : 1,000 ml = x &#181;moles : 0.250 ml 
	Note: The &#181;moles in 1,000 ml (number &#181;moles in 1 L) of are equivalent to the &#181;M concentration and x are the &#181;moles of nucleotide or nucleoside produced by 106 cells.
	<ol>
		<li>Lastly, convert the &#181;moles into nmoles consumed and/or produced by 106 CLL cells: 
		nmoles = &#181;moles x 1,000</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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.bdigitaluss.remotexs.co/files/ftp_upload/54124/54124fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><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&amp;rsquo;-diphosphate (ADP)</td><td>Sigma-Aldrich</td><td>A2754</td><td></td></tr><tr><td>adenosine 5&amp;rsquo;-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>&amp;alpha;,&amp;beta;-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><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>B&amp;uuml;rker cell counter</td><td>VWR</td><td>631-0920</td><td>hemocytometer</td></tr><tr><td>DynaMag-15 Magnet</td><td>Invitrogen</td><td>12301D</td><td>Dynal magnetic bead separator</td></tr><tr><td>microcentrifuge safe-lock tubes</td><td>Eppendorf</td><td>030-120-0086</td><td>1.5 ml</td></tr><tr><td>PET centrifuge tubes</td><td>Corning</td><td>430053/430304</td><td>15 &amp;ndash; 50 ml</td></tr><tr><td>Minisart RC4 syringe filters</td><td>Sartorius Stedim Biotech</td><td>17821</td><td>membrane 0.2 &amp;micro;m</td></tr><tr><td>short thread vials</td><td>VWR</td><td>548-0029</td><td>1.5 ml/glass</td></tr><tr><td>micro-inserts</td><td>VWR</td><td>548-0006</td><td>0.1 ml/glass</td></tr><tr><td>screw caps</td><td>VWR</td><td>548-0085</td><td>9 mm/PP blue</td></tr><tr><td>Atlantis dC18 Column</td><td>Waters</td><td>186001344</td><td>5 &amp;micro;m, 4.6 mm x 150 mm</td></tr><tr><td>Atlantis dC18 Guard Column</td><td>Waters</td><td>186001323</td><td>5 &amp;micro;m, 4.6 mm x 20 mm</td></tr><tr><td>Waters Alliance 2965 Separations Module</td><td>Waters</td><td></td><td>HPLC separation module</td></tr><tr><td>Waters 2998 Photodiode Array (PDA) Detector</td><td>Waters</td><td></td><td>UV detector</td></tr><tr><td>Waters Empower2 software</td><td>Waters</td><td></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 ...

hplc-based-assay-to-monitor-extracellular-nucleotidenucleoside

Universidad San Sebastian

Research

JoVE Journal

Chemistry

11.0K Views.  University of Turin. 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.

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

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.

This work describes an optimized methyl-CpG-binding domain (MBD) sequencing protocol and a computational pipeline to identify differentially methylated CpG-rich regions in chronic lymphocytic leukemia (CLL) patients.

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during ...

Genetics

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Cancer Research

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 ...

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 ...

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which are involved in many cell activities. Here, we describe a rapid and reliable method for the absolute qualification of nucleoside/nucleotide contents in plants. Briefly, 100 mg of homogenized plant material was extracted with 1 mL of extraction buffer (methanol, acetonitrile, and water at a ratio of 2:2:1). Later, the sample was concentrated five times in a freeze dryer and then injected into an HPLC-MS/MS. Nucleotides were separated on a porous graphitic carbon (PGC) column and nucleosides were separated on a C18 column. The mass transitions of each nucleoside and nucleotide were monitored by mass spectrometry. The contents of the nucleosides and nucleotides were quantified against their external standards (ESTDs). Using this method, therefore, researchers can easily quantify nucleosides/nucleotides in different plants.

A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...

Biochemistry

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Medicine

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Immunology and Infection

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Bromodeoxyuridine Pulse Labelling Assay: A Technique to Measure Cell Proliferation

Source: Welschinger R. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/t/52840">Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization.</a> J. Vis. Exp. (2015)
The video describes the stepwise pulse labeling assay of mammalian cells with bromodeoxyuridine (BrdU) for measuring cell proliferation. The BrdU uptake permits the temporal tracking of cells that were in the synthesis phase at a specific point in time, without requiring the cells to be synchronized.

Source: Welschinger R. et al. Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization. J. Vis. Exp. (2015)
...

JoVE Encyclopedia of Experiments

Leukemia

In vitro techniques & assays

Extracellular Purine Metabolism: An HPLC-based Assay to Measure Adenosine Generation and Metabolism in Leukemic Cells

Source: Serra S et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/t/54124">HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells.</a> J. Vis. Exp. (2016)
This video explains a quantitative reverse phase high-performance liquid chromatography (RP-HPLC) method to measure metabolism of adenosine and associated purines generated by consumption of AMP in extracellular environment of leukemic cancer cells. The protocol describes a sensitive, specific, reliable and reproducible 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.

Source: Serra S et al. HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells. J. Vis. ...