We thank the Lymphoid Malignancies Unit, Elisa ten Hacken, Lydia Scarf&#242;,&#160;Massimo Alessio, Antonio Conti, Angela Bachi, and Angela Cattaneo.
This project was supported by: Associazione Italiana per la Ricerca sul Cancro AIRC (Investigator Grant and Special Program Molecular Clinical Oncology &#8211; 5 per mille #9965)
C.S. and B.A designed the study, performed the experiments, analyzed the data and wrote the manuscript. M.T.S.B., U.R., F.B. P.R. assisted in writing the manuscript. P.G. and F.C.C. designed the study provided patients&#8217; samples and clinical data.

NOTE: All tissue samples were obtained with approval of the institutional review board of San Raffaele Hospital (Milan, Italy).
1. Human Tissue Samples and Cell Purification (Figure 1)
NOTE: Leukemic lymphocytes were obtained from the Peripheral Blood (PB) of CLL patients, diagnosed according to Mulligan et al. 9
1.1) Leukemic Cell Separation from PB
<ol>
	<li>Collect PB in vacutainer blood collection tubes with Sodium Heparin.</li>
	<li>Transfer blood into a 15 ml tube and add human B-cell enrichment cocktail at 50 &#956;L/mL of whole blood. Mix well and incubate 20 min at room temperature.</li>
	<li>Dilute sample with an equal volume of PBS + 2% FBS, mix gently and carefully overlay blood over the Ficoll, making sure not to break the interface. Use at least 1 part of Ficoll to 2 parts of the diluted sample (i.e. 4 ml Ficoll + 10 ml blood in a 15 ml tube).</li>
	<li>Centrifuge at 400 x g, at room temperature (20 &#176;C) for 20 min with no brake. Mononuclear cells will be at the interface.</li>
	<li>Carefully aspirate the interface to recover the cells and place into a new tube. Wash cells once with PBS and centrifuge at 300 x g, 4 &#176;C in the dark&#160;for 5 min. The pellet will be at the bottom.</li>
	<li>Discard the supernatant. Resuspend the pellet in the remaining supernatant by flicking the tube back and forth with fingers. Dilute the pellet with 10 ml complete RPMI medium (RPMI 1640 supplemented with 10% Fetal Calf Serum and 15 mg/ml gentamicin) and count cells using the Trypan Blue exclusion method.</li>
</ol>
1.2) Purity Evaluation
NOTE: Purity of all preparations needs to be always above 99%.
<ol>
	<li>Incubate 100 &#956;l&#160;of purified cell suspension with the following antibodies (commercially available; see Table of Materials): CD3FITC (5 &#181;l/test), CD14PE (5 &#181;l/test), CD19ECD (6 &#181;l/test), CD16-CD56PC5 (2 &#181;l/test), CD5PC7 (4 &#181;l/test), for 20 min at 4 &#176;C in a FACS tube.</li>
	<li>Wash the sample with 4 ml of PBS (centrifuge for 5 min at 300 x g) and check the purity by flow cytometry.Check in the plot the % of CLL cells (&#62; 99%) which co-express CD19 and CD5 on their surface as shown in Figure 1(6). Ensure that the preparations are virtually devoid of NK, T lymphocytes and monocytes by checking the % of CD3, CD14 and CD16-56 in the plot, which should be near zero.</li>
</ol>
1.3) Samples Storage
<ol>
	<li>For proteomic studies, collect 25 x 106 cells in a 1.5 ml tube, centrifuge cells at 300 x g for 10 min, discard the supernatant and lyophilize pellets for 4 hr. Keep at -80 &#176;C until use.</li>
	<li>For validation assays, resuspend cells in complete RPMI (quantity depends on the further assay that is chosen) and proceed with Step 4.</li>
</ol>
2. Two-dimensional Electrophoresis (2-DE) (Figure 2)
2.1) Isoelectrofocusing (IEF)
<ol>
	<li>Solubilize CLL cells pellets with a final volume of 380 &#956;l 2-DE buffer (344 &#956;l of a RBthio solution (9 M Urea, 10 mM Tris, 4% CHAPS), 30 &#956;l of 65mM DTT, 6 &#956;l of 2% IPG buffer ampholine pH 4-7).</li>
	<li>Apply protein samples (1 mg) to 18 IPG strips (pH 4-7) and perform IEF following standard protocol as described by Conti et al. 10</li>
	<li>Equilibrate strips in 2 ml of Equilibration Buffer (EB: 50 mM pH 8.8 Tris-HCl buffer containing 6 M urea, 30% glycerol, 2% SDS), supplemented with 2% of fresh DTT, for 15 minutes at RT. Discard EB+DTT and add 2 ml of EB supplemented with 2.5% Iodoacetamide. Incubate for 15 min at RT on a rocking platform.</li>
</ol>
2.2) SDS-PAGE
<ol>
	<li>Dry the strips on a paper without touching the gel to remove the excess of EB. Load each strip on top of a 9-16% gradient SDS-PAGE gel. Add a small volume of SDS-Electrophoresis Buffer (Tris 25 mM, Glycine 192 mM, SDS 0,1% w/v) on top of the gel to facilitate loading of the strip.</li>
	<li>Seal the gel by adding ~5 ml of agar solution (0.8%) to prevent floating of the strip, then assemble the electrophoretic unit and fill with SDS-Electrophoresis buffer. Perform the run at 60 mA/gel for 4 hr at 4 &#176;C.</li>
	<li>Remove the gel from the electrophoretic unit and place it in the proper staining box.</li>
</ol>
2.3) Silver Stain for Preparative Gel
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>FIXER</td>
			<td>50% Methanol 12% Acetic Acid 
			0,05% Formalin</td>
			<td>2 hr (or overnight)*</td>
		</tr>
		<tr>
			<td>WASH BUFFER</td>
			<td>&#160;35% Ehanol</td>
			<td>20 min (repeat the step three times)</td>
		</tr>
		<tr>
			<td>SENSITIZING</td>
			<td>&#160;0,02% Sodium Thiosulfate</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>WASH</td>
			<td>&#160;H2O</td>
			<td>5 min (repeat the step three times)&#160;</td>
		</tr>
		<tr>
			<td>SILVER NITRATE</td>
			<td>&#160;0,2% Silver Nitrate 0,076% Formalin</td>
			<td>20 min</td>
		</tr>
		<tr>
			<td>WASH</td>
			<td>&#160;H2O</td>
			<td>1 min (repeat the step twice)&#160;</td>
		</tr>
		<tr>
			<td>DEVELOPER</td>
			<td>6% Sodium Carbonate 0,0004% Sodium Thiosulfate 
			&#160;0,05% formalin</td>
			<td>Until the staining is sufficient</td>
		</tr>
		<tr>
			<td>STOP</td>
			<td>50% Methanol</td>
			<td>30 min</td>
		</tr>
		<tr>
			<td></td>
			<td>* 2 hr is the minimum time required for protein fixation&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1.
NOTE: Protein spots can be visualized by staining gels with MS compatible silver stain11. All the solutions needed for silver staining are listed in Table 1.
<ol>
	<li>Prepare about 200 ml of each solution/gel and proceed as indicated in Table 1. Carefully add each solution to the staining box containing the gel. Perform all incubations on a rocking platform.</li>
	<li>After the staining, remove the Stop Solution and leave the gel in a solution of 1% Acetic Acid until acquisition.</li>
</ol>
2.4) Gel Acquisition and Analysis
<ol>
	<li>Acquire images at high resolution using a densitometer within 3 days from the staining.</li>
	<li>Analyze 2-D protein patterns by using software for 2-DE gels. 
	NOTE: The software allows a fast and reliable image comparison.
	<ol>
		<li>Create a project by choosing &#8220;create a new project&#8221; from the contextual menu. Create a folder and import the gels by choosing &#8220;import gel images&#8221; with .tif, .png. or .gel extension.</li>
		<li>Once the gels are imported and added to the workspace, perform an automated spot detection by selecting the gels for spot detection and choosing &#8220;edit &#62; spot&#62; detect&#8221; in the menu.</li>
		<li>Next, perform a background subtraction by selecting from the menu &#8220;subtract background&#8221;. Note: After that it is possible to compare multiple gels and to detect differences or similarities by quantitative and/or qualitative analysis.</li>
	</ol>
	</li>
</ol>
3. Protein Identification by MALDI-TOF MS analysis (Figure 3)
3.1) Protein Digestion
<ol>
	<li>Rinse the gel with water and excise spots of interest from gels either by manual (cutting with a clean scalpel) or by automated excision.</li>
	<li>Wash gel particles with water (100-150 &#956;l), spin down (30 sec at 400 x g) and remove the liquid.</li>
	<li>Add an equal volume (depending on the gel volume) of CH3CN to the gel pieces and wait for 10-15 min until the gel pieces shrink. Dry the gel particles in a vacuum centrifuge.</li>
	<li>Add 75-100 &#181;l of 10 mM Dithioerythritol diluted in 50 mM NH4HCO3 and incubate for 30 min at 56 &#176;C (Reduction Step). Shrink again the gel pieces with CH3CN as described in Step 3.1.3.</li>
	<li>Proceed with alkylation by adding 75-100 &#181;l of a solution 55 mM Iodoacetamide diluted in 50 mM NH4HCO3 and incubate for 20 min at room temperature in the dark.</li>
	<li>Shrink the gel pieces once more with CH3CN using enough volume to cover the gel pieces as described in Step 3.1.3. Dry in a vacuum centrifuge.</li>
	<li>Rehydrate the particles in a buffer containing 25 mM NH4HCO3, 5 mM CaCl2,and 12,5 ng/&#956;l of trypsin at 4 &#176;C for 20 min. Use enough volume of buffer to cover the gel pieces. Remove the un-absorbed supernatant from the gel pieces and add 25 mM NH4HCO3, 5 mM CaCl2 without trypsin to cover the gel.</li>
	<li>Leave samples at 37 &#176;C at least 3 hr (the minimum time required for peptide digestion) to overnight.</li>
</ol>
3.2) Mass Spectrometry Analysis (Dried droplet technique)
<ol>
	<li>Spot 1 &#956;l aliquot of each sample onto a stainless steel MALDI plate and mix with 1 &#181;l of &#945;-cyano-4-hydroxycinnamic acid as matrix, prepared in 50% CH3CN and 0.1% TFA.</li>
	<li>Obtain mass spectra on a MALDI&#8211;TOF mass spectrometer. Accumulate about 200 spectra per spot, adjusting laser intensity to prevent signal saturation or to increase the signal. Process spectra via Data Explorer software as described below:
	<ol>
		<li>Import the .dat file and perform the baseline correction by choosing &#8220;process &#62; baseline correction&#8221;. Calibrate masses using the trypsin autolysis products and matrix peaks by choosing &#8220;process &#62; mass calibration &#62; manual calibration; select peaks close to m/z 855.1 and 2163.1 by left dragging and select the corresponding reference masses, then click &#8220;plot&#8221; and &#8220;apply calibration&#8221; in the manual calibration window.</li>
		<li>Adjust the threshold for peak detection (&#8220;peak &#62;peak detection&#8221;), duplicate the active trace (&#8220;display &#62;duplicate active trace&#8221;), select the new trace and perform the deisotoping (&#8220;peak &#62;deisotoping&#8221;). Using the macro &#8220;getpeaklist&#8221;, generate a list of masses to be used for the identification. If required, manually remove noise signals picked as peaks to clean the list and get a better identification.</li>
	</ol>
	</li>
	<li>Identify proteins by searching a comprehensive non-redundant protein database using ProFound and Mascot as search engines12.
	<ol>
		<li>Use the following parameters in all searches: one missed cleavage per peptide, methionine oxidation as variable modification, cysteine carbamidomethylation as fixed modification and an initial mass tolerance of 50 ppm.</li>
	</ol>
	</li>
</ol>
4. Cytoskeletal Activity Assays (Figure 4)
NOTE: Resuspend cells purified in Step 1 in complete RPMI (106 cells/200 &#956;l).
4.1) Migration Assay. This test allows quantification of the migratory capacity of the analyzed cells (lymphocytes ). Use a transwell chamber of 6.5 mm diameter and 5.0 &#956;m pore size and perform the assay in triplicate. Optimize pore size and migration time (Step 4.1.3) in the case of different cell types.
<ol>
	<li>Prepare the lower chamber by adding 600 &#956;l of complete RPMI with or without the specific stimuli (i.e. SDF-1) to measure the induced and the spontaneous migration respectively.</li>
	<li>Put the upper chamber on it and let the system equilibrate for 30 minutes at 37 &#176;C in the incubator.</li>
	<li>Seed 106 cells/200 &#956;l in the upper chamber (total cell number) and incubate the plate for 4 hr at 37 &#176;C in the incubator.</li>
	<li>Remove the upper chamber, collect the media in the lower chamber and wash the lower chamber once with 1 ml PBS. Collect the PBS in the tube.</li>
	<li>Centrifuge 5 minutes at 300 x g, discard the supernatant and resuspend in 500 &#956;l PBS.</li>
	<li>By flow cytometry, count the number of migrated cells after 1 min of acquisition. In case of an isolated cell population it is not necessary to add any surface antibody. Check the number of cells in a bi-dimensional dot plot considering the side scatters of the cells and the number of events visualized in 1 min, which is the number to use for calculation.</li>
	<li>Calculate the Migration Index (MI) as: 
	<img alt="Equation 1" fo:content-width="3.5in" src="/files/ftp_upload/51942/51942eq1.jpg" /></li>
</ol>
4.2) Adhesion assay. This assay allows measuring the adhesion capacity of the cells. Perform the assay in triplicate.
<ol>
	<li>Pre-coat 96-wells plate (flat-bottom) with 50 &#956;l/well of either 2% BSA-PBS (as background) or 1 &#956;g ICAM-1 diluted in PBS, overnight at 4 &#176;C.</li>
	<li>Wash the plate twice with PBS and block non-specific sites by adding 2% BSA-PBS (100 &#956;l/well) for 1 hr at 37 &#176;C.</li>
	<li>In the meantime resuspend cells at 5 x 106/ml in complete RPMI, label them with 1 mM CMFDA-green cell tracker and incubate 30 min at 37 &#176;C.</li>
	<li>Add 1 x 106 cells/well to the pre-coated wells after discarding the blocking solution (Step 4.2.2) and incubate 1 hr at 37 &#176;C.</li>
	<li>Quantify adhesion by using an ELISA reader (Excitation filter: 485 nM, Emission filter: 535 nM). Perform a first measurement on the total input (INPUT).</li>
	<li>Wash the plate gently with 2% BSA-PBS (200 &#956;l/well) 4 times (x). Perform a reading of the plate after each washing step (Adhesionx).</li>
	<li>After background subtraction for each measurement, calculate specific adhesion (ADHESION): 
	<img alt="Equation 1" fo:content-width="2in" src="/files/ftp_upload/51942/51942eq2.jpg" /></li>
</ol>
4.3) Polymerization assay. This is a colorimetric assay that quantifies F-actin polymerization. Perform the assay in triplicate.
<ol>
	<li>Resuspend 1x10^6 cells in 100 &#956;l of pre-warmed complete RPMI and add the specific stimulus (i.e. &#945;-IgM 20 &#956;g/ml ) for 10 min.</li>
	<li>Stop the reaction with 400&#160;&#956;l of 4% paraformaldehyde and fix cells for 10 min at RT.</li>
	<li>Wash 3 times with PBS (centrifuge at 300 x g for 5 min, 4 &#176;C).</li>
	<li>Discard the supernatant and permeabilize cells with PBS-saponine 0.2% (200&#160;&#956;l) on ice for 2 min.</li>
	<li>Wash 3 times with PBS-saponine 0.1% (centrifuge at 100 x g for 5 min, 4 &#176;C), discard the supernatant and resuspend in 100&#160;&#956;l&#160;PBS-saponine 0.1%.</li>
	<li>Stain with Phalloidin-Alexafluor 488 (3&#160;&#956;g/ml) for 30 min at room temperature in the dark.</li>
	<li>Wash 3 times with PBS-0.1% saponine. Discard the supernatant and resuspend in 300&#160;&#956;l PBS.</li>
	<li>Quantify the amount of F-actin by flow cytometry, calculating the Mean Florescence Intensity (MFI) of the Phalloidin. Analyze the generated data in a single dimension plot in order to produce a histogram based on the fluorescent intensity, the MFI value is shown in the graph report.</li>
	<li>Measure F-actin polymerization (&#8710; F-actin) as: 
	<img alt="Equation 1" fo:content-width="2in" src="/files/ftp_upload/51942/51942eq3.jpg" /></li>
</ol>

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The authors have nothing to disclose.

The aim of this manuscript is to describe an optimized protocol for the identification of molecules involved in the pathogenesis of CLL, even if this approach can be extended to other diseases and/or other sample types16-18. By comparing the proteomic profiles of 2 subsets of CLL patients, good vs bad prognosis19, we demonstrated that they differ in the phosphorylation status of HS1 and that its activation affects the migration and adhesion capacity of leukemic cells.
<p class="jove_con...

Chronic Lymphocytic Leukemia (CLL), the most common adult leukemia in the Western countries, derives from the accumulation of monoclonal neoplastic CD5+ B lymphocytes and is clinically very heterogeneous. It may be present as a pre-leukemic form, defined as monoclonal B-cell lymphocytosis (MBL) 1,2,3. In other cases the disease can appear either with an indolent clinical course, that can remain stable for years before needing treatment, or as a progressive chemorefractory disease with dismal prognosis despite therapy. Finally, it may progress into a frequently lethal high grade lymphoma (Richter&#8217;s Syndrome-RS) 2,3,4. Patients are usually classified in two main subsets: good and bad prognosis, based on a set of prognostic factors that provides complementary information on predictors of disease outcome and survival. Clinical heterogeneity likely reflects biological heterogeneity. A number of genetic, microenvironmental and cellular factors have been shown to concur to disease pathogenesis though no unifying mechanisms have been found 5. In detail, several studies have demonstrated that differences in the clinical course of the disease can be partially explained by the presence (or the absence) of some biological markers that have a prognostic value 6,7,8. These data demonstrated that a better understanding of the disease biology could provide additional hints for the clinical management of the pathology, by the identification of both prognostic markers and therapeutic targets.
The goal of the present paper is to show how a combination of different proteomic techniques can be used for the identification of proteins involved in CLL onset and progression. Our approach demonstrates that it is possible to join together basic proteomic and clinical data5.
In the present workflow, primary leukemic cells from selected patients (good vs bad prognosis) are isolated and cell lysates are then used to obtain proteomic maps. 2DE allows the characterization of the proteomic profile of a cell population, including post-translational modifications, thus giving indirect information on the biological activity of each protein. Whole cell lysates are resolved by a first dimension run based on the isoelectric point, followed by a second dimension run on a polyacrylamide gel that resolves proteins based on their molecular weight. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (both in terms of abundance and post-translational modifications) as spots on the gel that can be cut and analyzed by Mass Spectrometry. The role of each candidate can be then exploited by different assays.
This approach, restricted to CLL in the current manuscript, can be easily expanded to other diseases/samples, thus providing information about the proteomic/biological differences between two groups (i.e. pathologic vs normal, stimulated vs unstimulated, wild-type vs knockdown).

<table border="0" cellpadding="0" cellspacing="0" width="796">
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		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Name of Material/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Acetonitril</td>
			<td style="width:160px;">MERCK</td>
			<td style="width:111px;">61830025001730&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ammonium Bicarbonate</td>
			<td>SIGMA</td>
			<td style="width:111px;">A6141-500G</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:359px;">1,4-Dithioerythritol</td>
			<td>SIGMA</td>
			<td style="width:111px;">D9680-5G</td>
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			<td height="21" style="height:21px;width:359px;">Iodoacetamide</td>
			<td>SIGMA</td>
			<td style="width:111px;">I6125-25G</td>
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			<td height="21" style="height:21px;width:359px;">Calcium chloride dihydrate</td>
			<td>SIGMA</td>
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			<td height="21" style="height:21px;">&#945;-cyano-4-hydroxycinnamic acid</td>
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			<td height="21" style="height:21px;width:359px;">Trifluoroacetic acid</td>
			<td>SIGMA</td>
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			<td></td>
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			<td height="25" style="height:25px;width:359px;">ZipTip&#181;-C18</td>
			<td>MILLIPORE</td>
			<td>ZTC18M096</td>
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			<td height="21" style="height:21px;width:359px;">RosetteSep Human B Cell Enrichment Cocktail</td>
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			<td>15064</td>
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			<td height="21" style="height:21px;width:359px;">PBS</td>
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			<td>ECB4004L</td>
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			<td>1114547</td>
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			<td height="21" style="height:21px;width:359px;">Trypan Blue</td>
			<td>SIGMA</td>
			<td>T8154</td>
			<td></td>
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			<td height="21" style="height:21px;width:359px;">CD19</td>
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			<td>082A07770</td>
			<td>ECD</td>
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			<td height="21" style="height:21px;width:359px;">CD5</td>
			<td>BECKMAN COULTER</td>
			<td>082A07754</td>
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			<td height="21" style="height:21px;width:359px;">CD3</td>
			<td>BECKMAN COULTER</td>
			<td>082A07746</td>
			<td>FITC</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD14</td>
			<td>BD</td>
			<td>345785</td>
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			<td height="21" style="height:21px;width:359px;">CD16</td>
			<td>BECKMAN COULTER</td>
			<td>082A07767</td>
			<td>PC5</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD56</td>
			<td>BECKMAN COULTER</td>
			<td>082A07789</td>
			<td>PC5</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ettan IPGphor 3 Isoelectric Focusing Unit</td>
			<td style="width:160px;">GE-Healthcare</td>
			<td>11-0033-64&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Urea</td>
			<td>SIGMA</td>
			<td>U6504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Tris-Hcl Buffer</td>
			<td>BIO-RAD</td>
			<td>161-0799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CHAPS</td>
			<td>SIGMA</td>
			<td>C2020-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">DTT</td>
			<td>SIGMA</td>
			<td>D9163-5G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">IPGstrips</td>
			<td>GE-Healthcare</td>
			<td>17-1233-01&#160;</td>
			<td>Linear pH 4-7 18cm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">IPGbuffer</td>
			<td>GE-Healthcare</td>
			<td>17-6000-86</td>
			<td>pH 4-7</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Glycerol</td>
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			<td>G6279</td>
			<td></td>
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			<td>165-3188</td>
			<td></td>
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			<td height="21" style="height:21px;width:359px;">SDS</td>
			<td>INVITROGEN</td>
			<td>NP0001</td>
			<td></td>
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			<td height="21" style="height:21px;width:359px;">Acrilamide</td>
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			<td>161-0156</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Agarosio</td>
			<td>INVITROGEN</td>
			<td>16500</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Methanol</td>
			<td>SIGMA</td>
			<td>32213</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Acetic Acid</td>
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			<td>631000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Formalin</td>
			<td>BIO-OPTICAL</td>
			<td>05-K01009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ethanol</td>
			<td>VWR</td>
			<td>9832500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium Thiosulfate</td>
			<td>FLUKA</td>
			<td>72049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Silver Nitrate</td>
			<td>FLUKA</td>
			<td>85228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular Dynamics Personal SI Laser Densitometer</td>
			<td>Amersham Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageMaster 2D Platinum 5.0</td>
			<td>Amersham Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium Carbonate</td>
			<td>MERCK</td>
			<td>A0250292</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">MALDI-TOF Voyager-DE STR</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Data Explorer software (version 3.2)&#160;</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">transwell chambre 6.6 mm diameter</td>
			<td>CORNING</td>
			<td>3421</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">RPMI</td>
			<td>EUROCLONE</td>
			<td>ECB2000L</td>
			<td>WITH L-GLUTAMINE</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Gentamicin</td>
			<td>SIGMA</td>
			<td>G1397</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">SDF-1</td>
			<td>PREPROTECH</td>
			<td>300-28A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Flat-bottom 96-well plate</td>
			<td>BECTON DICKINSON</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">BSA</td>
			<td>SANTA CRUZ</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">ICAM-1</td>
			<td>R&#38;D Systems</td>
			<td>720-IC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CMFDA</td>
			<td>Life Thechnology</td>
			<td>C7025</td>
			<td>Green</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">IgM</td>
			<td>SOUTHERNBIOTECH</td>
			<td>2020-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Paraformaldehyde</td>
			<td>SIGMA</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Saponine</td>
			<td>SIGMA</td>
			<td>S-7900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Phalloidin&#160;</td>
			<td>Life Thechnology</td>
			<td>A12379</td>
			<td>Alexa fluor 488</td>
		</tr>
	</tbody>
</table>

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.

We isolated primary leukemic B cells form PB of CLL patients and we analyzed proteomic maps. Samples (n = 104) were grouped in two main subsets based on the clinical features of each patient (bad prognosis vs good prognosis) and comparative analysis of 2DE gels was performed.
The analysis allowed identification of spots differentially expressed between the two subsets (in term of presence/absence or shift on the gel, implying changes in post-translational modifications; n &#8776; 16)....

Watch this Scientific Journal Video about From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia at JoVE.com

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

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 ...

from-2de-gel-spot-to-protein-function-lesson-learned-from-hs1-chronic

Research

JoVE Journal

Medicine

13.6K Views.  IRCCS, San Raffaele Scientific Institute.  Qui si descrive un protocollo che le coppie due tecniche di proteomica, cioè 2-dimensionale elettroforesi (2DE) e spettrometria di massa (MS), per identificare differenzialmente espressi / proteine ​​modificate post-traduzionali tra due o più gruppi di campioni elementari. Questo approccio, insieme a esperimenti funzionali, permette l'identificazione e la caratterizzazione di marcatori prognostici / bersagli terapeutici. 

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

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

Utilizing Repetitive Transcranial Magnetic Stimulation to Improve Language Function in Stroke Patients with Chronic Non-fluent Aphasia

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this experiment, we demonstrate the administration of low-frequency repetitive TMS (rTMS) to an optimal stimulation site in the right hemisphere in patients with chronic non-fluent aphasia. A battery of standardized language measures is administered in order to assess baseline performance. Patients are subsequently randomized to either receive real rTMS or initial sham stimulation. Patients in the real stimulation undergo a site-finding phase, comprised of a series of six rTMS sessions administered over five days; stimulation is delivered to a different site in the right frontal lobe during each of these sessions. Each site-finding session consists of 600 pulses of 1 Hz rTMS, preceded and followed by a picture-naming task. By comparing the degree of transient change in naming ability elicited by stimulation of candidate sites, we are able to locate the area of optimal response for each individual patient. We then administer rTMS to this site during the treatment phase. During treatment, patients undergo a total of ten days of stimulation over the span of two weeks; each session is comprised of 20 min of 1 Hz rTMS delivered at 90% resting motor threshold. Stimulation is paired with an fMRI-naming task on the first and last days of treatment. After the treatment phase is complete, the language battery obtained at baseline is repeated two and six months following stimulation in order to identify rTMS-induced changes in performance. The fMRI-naming task is also repeated two and six months following treatment. Patients who are randomized to the sham arm of the study undergo sham site-finding, sham treatment, fMRI-naming studies, and repeat language testing two months after completing sham treatment. Sham patients then cross over into the real stimulation arm, completing real site-finding, real treatment, fMRI, and two- and six-month post-stimulation language testing.

We explore the use of repetitive transcranial magnetic stimulation (rTMS) to improve language abilities in patients with chronic stroke and non-fluent aphasia. After identifying a site in the right frontal gyrus for each patient that responds optimally to stimulation, we target this site during ten days of rTMS treatment.

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this ...

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e.&#160;samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.

Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates&#160;and analyzed for a variety of biomarkers.

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

A Protocol to Acquire the Degenerative Tenocyte from Humans

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical activity and longer life expectancy. Despite its increasing prevalence, the underlying pathogenesis still remains unclear, and treatment is generally symptomatic. Recently, numerous therapeutic options, including growth factors, stem cells, and gene therapy, were investigated in hopes of enhancing the healing potency of the degenerative tendon. However, the majority of these research studies were conducted only on animal models or healthy human tenocytes. Despite some studies using pathological tenocytes, to the best of our knowledge there is currently no protocol describing how to obtain human degenerative tenocytes. The aim of this study is to describe a standard protocol for acquiring human degenerative tenocytes. Initially, the tendon tissue was harvested from a patient with lateral epicondylitis during surgery. Then biopsy samples were taken from the extensor carpi radialis brevis tendon corresponding to structural changes observed at the time of surgery. All of the harvested tendons appeared to be dull, gray, friable, and edematous, which made them visually distinct from the healthy ones. Tenocytes were cultured and used for experiments. Meanwhile, half of the harvested tissues were analyzed histologically, and it was shown that they shared the same key features of tendinopathy (angiofibroblastic dysplasia or hyperplasia). A secondary analysis by immunocytochemistry confirmed that the cultured cells were tenocytes with the majority of the cells having positive stains for mohawk and tenomodulin proteins. The qualities of the degenerative nature of tenocytes were then determined by comparing the cells with the healthy control using a proliferation assay or qRT-PCR. The degenerative tenocyte displayed a higher proliferation rate and similar gene expression patterns of tendinopathy that matched previous reports. Overall, this new protocol might provide a useful tool for future studies of tendinopathy.

In vitro use of degenerative tenocytes is essential when investigating the efficacy of novel treatment on tendinopathy. However, most research studies use only the animal model or a healthy tenocyte. We propose the following protocol to isolate human degenerative tenocytes during surgery.

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...