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.remotexs.ntu.edu.sg/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>

<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 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.remotexs.ntu.edu.sg/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 ...

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

Microsoft Bing

Research

JoVE Journal

Chemistry

10.9K 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

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

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, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

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 engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

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 identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by 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 of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

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

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

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 structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

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. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

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;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

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 phase of drug developing process because they are genetically human and recapitulate the interplay among tissues. In this study, the human intestinal barrier (emulated by a co-culture of Caco-2 and HT-29 cells) and the liver equivalent (emulated by spheroids made of differentiated HepaRG cells and human hepatic stellate cells) were integrated into a two-organ chip (2-OC) microfluidic device to assess some acetaminophen (APAP) pharmacokinetic (PK) and toxicological properties. The MPS had three assemblies: Intestine only 2-OC, Liver only 2-OC, and Intestine/Liver 2-OC with the same media perfusing both organoids. For PK assessments, we dosed the APAP in the media at preset timepoints after administering it either over the intestinal barrier (emulating the oral route) or in the media (emulating the intravenous route), at 12 &#181;M and 2 &#181;M respectively. The media samples were analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Organoids were analyzed for gene expression, for TEER values, for protein expression and activity, and then collected, fixed, and submitted to a set of morphological evaluations. The MTT technique performed well in assessing the organoid viability, but the high content analyses (HCA) were able to detect very early toxic events in response to APAP treatment. We verified that the media flow does not significantly affect the APAP absorption whereas it significantly improves the liver equivalent functionality. The APAP human intestinal absorption and hepatic metabolism could be emulated in the MPS. The association between MPS data and in silico modeling has great potential to improve the predictability of the in vitro methods and provide better accuracy than animal models in pharmacokinetic and toxicological studies.

We exposed a microphysiological system (MPS) with intestine and liver organoids to acetaminophen (APAP). This article describes the methods for organoid production and APAP pharmacokinetic and toxicological property assessments in the MPS. It also describes the tissue functionality analyses necessary to validate the results.

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 of their functions in nature and potential human benefits (e.g., for drug discovery applications) are desired. This protocol describes the combination of genome mining (GM) and molecular networking (MN), two contemporary approaches that match gene cluster-encoded annotations in whole genome sequencing with chemical structure signatures from crude metabolic extracts. This is the first step towards the discovery of new natural entities. These concepts, when applied together, are defined here as MS-guided genome mining. In this method, the main components are previously designated (using MN), and structurally related new candidates are associated with genome sequence annotations (using GM). Combining GM and MN is a profitable strategy to target new molecule backbones or harvest metabolic profiles in order to identify analogues from already known compounds.

A mass spectrometry-guided genome mining protocol is established and described here. It is based on genome sequence information and LC-MS/MS analysis and aims to facilitate identification of molecules from complex microbial and plant extracts.

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