This work was supported by the Institute National du Cancer (INCa-DGOS-4664).

1. Neutrophil Isolation and Co-culture with Primary Leukemic Cells
NOTE: Procedures were conducted under the approval of Lyon Hospital Ethics committee with all patients signing informed consent.
<ol>
	<li>Isolation of Primary Leukemic Cells and Neutrophils
	<ol>
		<li>Collect tubes of peripheral blood on EDTA (1.8 mg EDTA per milliliter of blood) from patients diagnosed with chronic lymphocytic leukemia (CLL).</li>
		<li>Add each 15 ml of blood to a sterile 50 ml tube and dilute with 15 ml RPMI (dilution 1:1), then carefully and slowly add 15 ml of density gradient solution to the bottom of the tube without mixing the phases. Ensure that the density gradient solution is at RT&#160;for the preparation.</li>
		<li>Centrifuge at 300 x g for 35 min at RT and without brake. The blood should separate into four distinct phases as shown in Figure 1A, top to bottom: platelet and plasma, mononuclear cells (white ring), density gradient solution, granulocytes and erythrocytes.</li>
		<li>Collect the white ring which represents the primary leukemic cells with a plastic Pasteur pipette and transfer to a new 50 ml tube.
		<ol>
			<li>Fill the tube with PBS (contains calcium and magnesium) up to 50 ml in total and centrifuge at 300 g for 10 min at RT.</li>
			<li>Resuspend the pellet with 5 ml PBS then add PBS up to 50 ml in total and centrifuge at 300 g for 10 min at RT.</li>
			<li>Resuspend the pellet with complete RPMI medium (RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin) for counting using cell viability counter.</li>
		</ol>
		</li>
		<li>Aspirate the upper phases leaving the granulocytes and erythrocytes phase and add PBS up to 25 ml then add 3% dextran in 0.9% NaClup to 50 ml in total. Mix the tubes 10 times and keep them for 30 min at RT without mixing.</li>
		<li>Collect the upper RBC-poor neutrophil layer and place them into a clean sterile 50 ml tube and centrifuge the tubes at 300 g for 10 min at RT. Resuspend each pellet with 5 ml PBS then add red cell lysis buffer up to 50 ml in total.</li>
		<li>Keep the tubes in dark for 15 min at RT then centrifuge at 500 g for 10 min at RT. Wash the pellets with PBS (add PBS up to 50 ml in total) then centrifuge at 500 g for 10 min at RT.</li>
		<li>Resuspend the pellets with complete RPMI medium for counting using cell viability counter. Keep 3 x 105 of each cell type in 50 &#181;l PBS-FBS (4%) in 5 ml plastic tubes to determine the purity of their isolation using flow cytometry.</li>
	</ol>
	</li>
	<li>Analyze the Morphological Appearances of the Isolated Cell Populations
	<ol>
		<li>To do so, resuspend each 3 x 104 neutrophils and 3 x 104 mononuclear cells in 150 &#181;l complete RPMI medium. Assemble the glass slides, filter cards, and sample chambers in the cytospin centrifuge, ensuring that the centrifuge is well balanced.</li>
		<li>Place each cell type into sample chamber and cyto-centrifuge at 750 x g for 10 min at RT Remove the slides, filter cards, and sample chambers from the centrifuge. Disassemble carefully, so as not to damage the cells on the slide. Discard filter cards and sample chambers.</li>
		<li>Stain the cells using Giemsa staining kit and keep the slides for 1 hr&#160;to dry. Examine the cells by microscope with 100X magnification.</li>
	</ol>
	</li>
	<li>Test the Purity of Isolated Cells by FACS
	<ol>
		<li>Label the isolated primary leukemic cells with anti-human CD19 antibody conjugated to APC (5 &#181;l/106 cells). Label the purified neutrophils with mixture of anti-human antibodies listed in Table 1 (5 &#181;l/106 cells). Incubate the cells in dark for 30 min at 4 oC.</li>
		<li>Wash the cells with 500 &#181;l PBS-FBS (4%) the centrifuge the tubes at 300 g for 5 min at RT.</li>
		<li>Resuspend each pellet in 200 &#181;l PBS-FBS (4%).</li>
		<li>Analyze on a flow cytometer using the following optical configuration: 488 nm laser: FSC-A (488 nM), SSC-A (488/10BP), FITC (530/30 BP), PerCP-Cy5.5 (695/40 BP), PE (575/26 BP); 633 nm laser: APC (660/20 BP), APC-Cy7 (780/60 BP). Ensure the color compensation.</li>
	</ol>
	</li>
	<li>Coculture of Primary Leukemic Cells with Autologous Neutrophils
	<ol>
		<li>Seed 2 x 105 cells/ml of primary leukemic cells alone or with autologous neutrophils at ratio 1:10 in complete RPMI medium. To do so, adjust cell numbers by diluting cell suspensions and add 2 x 105 cells/ml of primary leukemic cells to 2 x 106 cells/ml neutrophils. Mix carefully.</li>
		<li>Add 25 &#181;M of Bruton&#39;s tyrosine kinase (Btk) inhibitor (ibrutinib) to the cells. Incubate for 24 hr at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>Cell Harvest and FACS Analysis
	<ol>
		<li>Collect the cells in 5 ml plastic tubes for FACS analysis and centrifuge the tubes at 300 g for 5 min at RT. Wash the pellets with 1 ml PBS-FBS (4%) then centrifuge at 300 g for 5 min at RT.</li>
		<li>Resuspend pellets in Annexin V and PI using commercial kit and incubate the cells in dark for 10 min at RT. Analyze on a flow cytometer using the gating strategy of Figure 2A and the following optical configuration: 488 nm laser: FSC-A (488 nM), SSC-A (488/10BP), FITC (530/30 BP), PI (610/20 BP). Ensure the color compensation.</li>
	</ol>
	</li>
</ol>
2. Differentiation of HL60 Cells along the Granulocytic Pathway and Their Coculture with RL Lymphoma B Cells in 3D Model
<ol>
	<li>Differentiation of Human Promyelocytic (HL60) Cells into Neutrophil-like Cells
	<ol>
		<li>Adjust HL60 cell number to 3 x 105cells/ml then add 1 &#181;M retinoic acid (RA) and 1.25% DMSO to induce their differentiation. Seed each 3 x 105 HL60 cells per well in 48-well plate then incubate for 48 hr at 37 &#176;C with 5% CO2.</li>
		<li>Later, collect the cells and centrifuge at 300 g for 5 min at RT. Resuspend the cells with complete RPMI medium for counting using cell viability counter.</li>
		<li>Then dilute cell suspension in complete RPMI medium to adjust HL60 cell number to 3 x 105 cells/ml and induce again their differentiation by adding 1 &#181;M retinoic acid (RA) and 1.25 % DMSO. Seed each 3 x 105 HL60 cells per well in 48-well plate and incubate for another 48 hr at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>Analysis of HL60 differentiation (HL60diff)
	<ol>
		<li>Collect the cells and centrifuge the tubes at 300 g for 5 min at RT. Resuspend the pellet with complete RPMI medium for counting using cell viability counter.</li>
		<li>To analyze the changes in cell-surface markers expression by flow cytometry. Place each 3 x 105 undifferentiated HL60 cells (from cell culture) and 3 x 105 HL60diff cells in 5 ml plastic tubes for FACS analysis and centrifuge the tubes at 300 g for 5 min at RT.</li>
		<li>Resuspend the pellets in 50 &#181;l PBS-FBS (4%). Label the cells with anti-human CD11b conjugated to AF700 or anti-human CD38 conjugated to APC (5 &#181;l/106 cells). Incubate the cells in dark for 30 min at 4 oC.</li>
		<li>Wash with 500 &#181;l PBS-FBS (4%) then centrifuge at 300 g for 5 min at RT. Resuspend the pellets in 200 &#181;l PBS-FBS (4%).</li>
		<li>Analyze on flow cytometer using the gating strategy of Figure 3A and the following optical configuration: 488 nm laser: FSC-A (488 nM), SSC-A (488/10BP); 633 nm laser: APC (660/20 BP), Alexa Fluor 700 (730/45 BP).</li>
		<li>To test the morphological changes in HL60diff, immobilize the cells onto glass slides for microscopic examination.
		<ol>
			<li>To do so, resuspend each 3 x 104 undifferentiated HL60 cells (from cell culture) and 3 x 104 HL60diff in 150 &#181;l complete RPMI medium. Assemble the glass slides, filter cards, and sample chambers in the cytospin centrifuge, ensuring that the centrifuge is balanced.</li>
			<li>Place each cell type into sample chamber and cyto-centrifuge at 1,500 rpm for 10 min at RT. Remove the slides, filter cards, and sample chambers from the centrifuge. Disassemble carefully, so as not to damage the cells on the slide. Discard filter cards and sample chambers.</li>
			<li>Stain the cells using Giemsa staining kit and keep the slides for 1 hr to dry. Examine the cells by microscope with 100X magnification.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>3-dimensional (3D) Culture 
	NOTE: Ensure that the materials used in this experiment are cold and the experiment is taking place on ice.
	<ol>
		<li>Resuspend each 5 x 104 RL cells, either alone or mixed with HL60diff at ratio RL:HL60diff 1:10, with 300 &#181;l basement membrane matrix using 1 ml pipette tip cut with a sterile scissor in order to widen the opening to about 2 to 3 mm. Avoid bubbles during this step.</li>
		<li>Seed each 300 &#181;l of cell suspension/well in 24-well plate. Avoid bubbles during this step. Incubate the plate for 30 min at 37 &#176;C with 5% CO2then add 1 ml complete RPMI medium for each well and incubate for 7 days at 37 &#176;C with 5% CO2. Change the medium every two days. Add 10 nM vincristine at day 5.</li>
	</ol>
	</li>
	<li>FACS Analysis
	<ol>
		<li>After 7 days of culture, aspirate the medium and wash each well with 1 ml ice-cold PBS twice. Add 3 ml/well of ice-cold PBS-EDTA (5 mM). Detach the gel from the bottom of the well by scraping using the bottom of 200 &#181;l pipette tip. Shake the plate gently on ice for 30 min.</li>
		<li>Transfer cell suspension into sterile 15 ml tube and shake the tubes gently on ice for another 30 min. Check for the appearance of homogeneous cell suspension. (If it&#39;s not the case, then shake the cells for longer time or add more PBS-EDTA).</li>
		<li>Centrifuge the tubes at 300 x g for 10 min at RT. Wash the pellets with PBS then centrifuge at 300 x g for 10 min at RT.</li>
		<li>Resuspend with PBS-FBS (4%) and label with anti-human CD19 antibody conjugated to PE-Cy7 and anti-human CD38 antibody conjugated to APC (5 &#181;l/106 cells). Incubate in dark for 30 min at 4 oC.</li>
		<li>Wash with 500 &#181;l PBS-FBS (4%) then centrifuge tubes at 300 g for 5 min at RT. Resuspend the cells with Annexin V and PI using commercial kit.</li>
		<li>Analyze on flow cytometer using the gating strategy of Figure 4A and the following optical configuration: 488 nm laser: FSC-A (488 nM), SSC-A (488/10BP), FITC (530/30 BP), PI (610/20 BP), PE-Cy7 (780/60 BP); 633 nm laser: APC (660/20 BP). Ensure the color compensation.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Described here, an effective, simple, fast and inexpensive protocol for the isolation of neutrophils from human blood with high purity using density gradient centrifugation approach and within the same step mononuclear cells are also separated and recovered. The isolated cell populations are &#8805;90% pure.
Several methods are available for neutrophil isolation from human blood. These include similar methods using discontinuous gradients11,12, or using commercial kits for neutrophi...

Innate immune cells constitute an essential proportion of the cells within the tumor microenvironment and have been associated with tumor malignancy in patients and animal models of cancer1. Recently, it has become more widely appreciated that chronic immune responses play critical roles in promoting tumor progression, metastasis and resistance to chemotherapies2. Macrophages are important innate immune cells that have been shown to directly regulate tumor cell response to chemotherapy 3,4. However, the role of neutrophils, key players in the innate immune system, in regulating tumor response to anti-cancer treatment is not known. The aims of these protocols are to use a fast and credible method to separate neutrophils from CLL patient&#39;s blood samples and to differentiate HL60 cells along the granulocytic pathway in order to study their role in regulating the sensitivity of lymphoma cells to anti-lymphoma agents.
Neutrophils are the most abundant cellular component of the innate immune system in blood 5 and act as a first line of defense against invading microorganisms 6. Neutrophils have an essential role in rising effective innate immune responses in addition to variable effector functions in several pathological conditions 7. Therefore, a fast and credible method to isolate neutrophils from other blood cells, such as density gradient separation method, is required for in vitro studies. Using this method for neutrophil isolation will facilitate further research on neutrophil-mediated immunological functions in vivo and ex vivo.
The ability to obtain pure populations of neutrophils is an important first step for the investigation of patients with immunological diseases 8. Density gradient separation method is an ideal technique in which a high yield of cells is obtained. The method involves the addition of density gradient solution in the bottom of a tube containing diluted human blood followed by centrifugation at 300 g for 35 min without break. The ring of the mononuclear cells appears at the interface and the neutrophils reside below the former. This method have significant advantages with respect to other available methods such as neutrophil isolation kits which are much more expensive9. In addition, isolating neutrophils from human blood by commercial kits using antibodies directed to a surface marker specific for human neutrophils, increase the risk of cell activation or differentiation. Density gradient separation method allows the isolation of neutrophils within a short period of time. Within the same step, mononuclear cells are also separated and recovered. It&#39;s a fundamental technique in which a high yield of pure cells is obtained in order to achieve functional integrity.
In order to mimic the tumor microenvironment, 3D experiments were performed. Given the short half-life of neutrophils in vitro, the 3D experiments with fresh human neutrophils are not conclusive. For this reason, promyelocytic (HL60) cells are induced to differentiate into neutrophil-like cells using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). Using differentiated HL60 cells (HL60diff) will prevent having different responses of neutrophils due to isolation from different donors.
In vitro 3D culture models represent an intermediate stage between in vitro 2D models and in vivo models. In 2D culture, the cells spread on plastic surface forming unnatural cell attachments to deposited proteins that are denatured on this synthetic surface. Conversely, the cells in 3D culture form natural cell-cell attachments since the cells and the extracellular matrix they synthesize are the natural material to which they are attached. For this reason, 3D co-culture models, especially between cancer cells and other cell types, have been very useful for indicating their contribution to tumor growth, angiogenesis, and metastasis. As a result, 3D cultures make the cell culture mimic the physiological conditions that exist in vivo 10.

<table><tbody><tr><td>RPMI 1640&amp;nbsp;</td><td>Gibco Invitrogen</td><td>21875-034</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco Invitrogen</td><td>10270-106</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS contains calcium and magnesium)</td><td>Gibco Invitrogen</td><td>14040-091</td><td></td></tr><tr><td>N-acetyl-L-alanyl-L-glutamine (L-Glutamine)</td><td>Life technologies</td><td>25030-024</td><td></td></tr><tr><td>Penicillin streptomycin (Pen Strep)</td><td>Life technologies</td><td>15140-122</td><td></td></tr><tr><td>Bruton&#039;s tyrosine kinase (Btk) inhibitor (Ibrutinib)&amp;nbsp;</td><td>CliniSciences</td><td>A3001</td><td></td></tr><tr><td>Vincristine&amp;nbsp;</td><td>EG labo</td><td></td><td></td></tr><tr><td>BD Matrigel basement membrane matrix&amp;nbsp;</td><td>BD Biosciences</td><td>354234</td><td>Put at 4 &lt;sup&gt;o&lt;/sup&gt;C overnight (O/N) before the day of the experiment</td></tr><tr><td>Red cell lysis buffer&amp;nbsp;</td><td>BD Biosciences</td><td>555899</td><td></td></tr><tr><td>Ficoll (Pancoll)</td><td>PAN Biotech</td><td>P04-60500</td><td></td></tr><tr><td>Dextran&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8906</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma-Aldrich</td><td>R2625</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Euromedex</td><td>S3014</td><td></td></tr><tr><td>Annexing V-FLOUS staining kit&amp;nbsp;</td><td>Roche</td><td>11&amp;nbsp;988&amp;nbsp;549&amp;nbsp;001</td><td></td></tr><tr><td>Kit RAL 555 Modified Giemsa staining kit</td><td>Cosmos Biomedical</td><td>CB361550-0000</td><td></td></tr><tr><td>LSRII flow cytometry</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Cytocentrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Leica DMR-XA microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Cellometer Auto T4 Cell Viability Counter</td><td>Nexcelom&amp;nbsp; Bioscience</td><td></td><td></td></tr></tbody></table>

neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents

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

Density gradient separation method described here provides primary leukemic cells and unstimulated neutrophils isolated from the blood of CLL patients. Figure 1A represents the different blood layers obtained after density gradient centrifugation (from top to bottom: platelets and plasma, white ring represents the mononuclear cells, density gradient solution, granulocytes and erythrocytes). Figure 1B and 1C show the differences in the morphological appear...

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

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

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

neutrophil-isolation-analysis-to-determine-their-role-lymphoma-cell

Research

JoVE Journal

Immunology and Infection

15.2K Views.  Cancer Research Center in Lyon. Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents. 

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

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved in various disease processes, such as autoimmune disease, thrombosis, and tumor metastases. Here, we show a detailed in vitro technique to detect NET activity during the trapping of free tumor cells, which grow after attachment to NETs. First, we collected low density neutrophils (LDN) from postoperative peritoneal lavage fluid from patients who underwent laparotomies. Short-term culturing of LDN resulted in massive NET formation that was visualized with green fluorescent nuclear and chromosome counterstain. After co-incubation of human gastric cancer cell lines MKN45, OCUM-1, and NUGC-4 with the NETs, many tumor cells were trapped by the NETs. Subsequently, the attachment was completely abrogated by the degradation of NETs with DNase I. Time-lapse video revealed that tumor cells trapped by the NETs did not die but instead grew vigorously in a continuous culture. These methods may be applied to the detection of adhesive interactions between NETs and various types of cells and materials.

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

Cancer Research

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

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

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

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

Assessment of the Metabolic Profile of Primary Leukemia Cells

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

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

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

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.

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 ...

Chemistry

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

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

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

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

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

Medicine

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

The Isolation and Characterization of Low- and Normal- Density Neutrophils from Whole Blood

Emerging research shows that the circulating neutrophil population in humans consists of diverse subtypes and should not be studied as a single population, as has been done historically. In particular, low-density and normal-density neutrophils (LDNs, NDNs) have been shown to have functionally and metabolically distinct profiles, a factor that must be considered when publishing neutrophil research. Here, we present a modified method for the untouched isolation and separation of LDNs and NDNs from whole blood.
The density gradient medium (1.135 g/mL) is combined at 9:10 with 10x PBS. Specific density gradients of 55%, 70%, and 81% are subsequently made by combining the 100% density gradient medium with 1x phosphate-buffered saline (PBS). Neutrophils isolated from 12 mL of peripheral whole blood obtained from consented donors using a negative selection-based magnetic isolation kit are resuspended in the 55% fraction. A volume of 3 mL of the 81% and 70% fractions is layered into a 15 mL tube, followed by the 55% fraction containing total neutrophils. The density gradients are then centrifuged at 720 x&#160;g for 30 min. Two distinct bands are obtained at the 55%/70% interface (LDNs) and 70%/81% interface (NDNs). The cells are carefully pipetted into separate tubes and washed using PBS. The purity of the isolated fractions is determined using flow cytometry. Both LDNs and NDNs were defined as CD14lo CD15+ SSChi by flow cytometry. Isolation purity was calculated at &#8805;93% of viable cells for both types.
This method provides a reliable and efficient approach for separating LDN and NDNs from peripheral blood, ensuring high purity and viability of the isolated cells. Enhancing the precision of neutrophil isolation facilitates more accurate downstream analyses of these distinct neutrophil subpopulations. These are critical for advancing our understanding of neutrophil heterogeneity and its implications in various physiological and pathological contexts.

Here, we provide a reliable approach for isolating low- and normal-density neutrophils from whole blood using magnetic isolation (negative selection) and discontinuous density gradient medium. It ensures untouched isolation of high-purity cells (&#8805;93%), facilitating accurate downstream analysis of neutrophil subpopulations, crucial for understanding their roles in health and disease.

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

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