Name: neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents
Uploaded: 2016-03-25T15:00:00
Description: Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

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 d...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+are+not+less+sensitive+than+other+blood+leukocytes+to+the+genomic+effects+of+glucocorticoids.">Neutrophils are not less sensitive than other blood leukocytes to the genomic effects of glucocorticoids.</a> PloS One. 7, 44606 (2012).</li><li>Dorward, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+advance:+autofluorescence-based+sorting:+rapid+and+nonperturbing+isolation+of+ultrapure+neutrophils+to+determine+cytokine+production.">Technical advance: autofluorescence-based sorting: rapid and nonperturbing isolation of ultrapure neutrophils to determine cytokine production.</a> J. Leukoc. Biol. 94, 193-202 (2013).</li><li>Swamydas, M., Lionakis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+Purification+and+Labeling+of+Mouse+Bone+Marrow+Neutrophils+for+Functional+Studies+and+Adoptive+Transfer+Experiments.">Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments.</a> J Vis Exp. , e50586 (2013).</li><li>Scaife, H., Woldehiwet, Z., Hart, C. A., Edwards, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaplasmaphagocytophilum+reduces+neutrophil+apoptosis+in+vivo.">Anaplasmaphagocytophilum reduces neutrophil apoptosis in vivo.</a> Infect. Immun. 71, 1995-2001 (2003).</li><li>Maianski, N. A., Maianski, A. N., Kuijpers, T. W., Roos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+of+neutrophils.">Apoptosis of neutrophils.</a> ActaHaematol. 111, 56-66 (2004).</li><li>Dalton, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HL-60+cell+line+was+derived+from+a+patient+with+FAB-M2+and+not+FAB-M3.">HL-60 cell line was derived from a patient with FAB-M2 and not FAB-M3.</a> Blood. 71, 242-247 (1988).</li><li>Hogan, C., Kajita, M., Lawrenson, K., Fujita, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+normal+and+transformed+epithelial+cells:+Their+contributions+to+tumourigenesis.">Interactions between normal and transformed epithelial cells: Their contributions to tumourigenesis.</a> Int. J. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessories+to+the+crime:+functions+of+cells+recruited+to+the+tumor+microenvironment.">Accessories to the crime: functions of cells recruited to the tumor microenvironment.</a> Cancer Cell. 3, 309-322 (2012).</li><li>Shojaei, F., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refractoriness+to+antivascular+endothelial+growth+factor+treatment:+role+of+myeloid+cells.">Refractoriness to antivascular endothelial growth factor treatment: role of myeloid cells.</a> Cancer Res. 68, 5501-5504 (2008).</li><li>Liang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+promote+the+malignant+glioma+phenotype+through+S100A4.">Neutrophils promote the malignant glioma phenotype through S100A4.</a> Clin Cancer Res Off J Am Assoc Cancer Res. 20, 187-198 (2014).</li><li>Teramukai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretreatment+neutrophil+count+as+an+independent+prognostic+factor+in+advanced+non-small-cell+lung+cancer:+an+analysis+of+Japan+Multinational+Trial+Organisation+LC00-03.">Pretreatment neutrophil count as an independent prognostic factor in advanced non-small-cell lung cancer: an analysis of Japan Multinational Trial Organisation LC00-03.</a> Eur. 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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 border="0" cellpadding="0" cellspacing="0" width="1161">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">RPMI 1640&#160;</td>
			<td style="width:157px;">Gibco Invitrogen</td>
			<td style="width:156px;">21875-034</td>
			<td style="width:535px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fetal bovine serum (FBS)</td>
			<td>Gibco Invitrogen</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Phosphate-buffered saline (PBS contains calcium and magnesium)</td>
			<td>Gibco Invitrogen</td>
			<td>14040-091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-acetyl-L-alanyl-L-glutamine (L-Glutamine)</td>
			<td>Life technologies</td>
			<td style="width:156px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin streptomycin (Pen Strep)</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bruton&#39;s tyrosine kinase (Btk) inhibitor (Ibrutinib)&#160;</td>
			<td style="width:157px;">CliniSciences</td>
			<td style="width:156px;">A3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vincristine&#160;</td>
			<td style="width:157px;">EG labo</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">BD Matrigel basement membrane matrix&#160;</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Put at 4 oC overnight before the day of the experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Red cell lysis buffer&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:156px;">555899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll (Pancoll)</td>
			<td>PAN Biotech</td>
			<td style="width:156px;">P04-60500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran&#160;</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>D8906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Retinoic acid</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:156px;">R2625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Bovine serum albumin (BSA)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>A7906</td>
			<td style="width:535px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:156px;">E5134</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sodium chloride (NaCl)</td>
			<td>Euromedex</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Annexing V-FLOUS staining kit&#160;</td>
			<td style="width:157px;">Roche</td>
			<td>11&#160;988&#160;549&#160;001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Kit RAL 555 Modified Giemsa staining kit</td>
			<td style="width:157px;">Cosmos Biomedical</td>
			<td style="width:156px;">CB361550-0000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSRII flow cytometry</td>
			<td>BD Biosciences</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Cytocentrifuge</td>
			<td style="width:157px;">Thermo Scientific</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMR-XA microscope</td>
			<td style="width:157px;">Leica Microsystems</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Cellometer Auto T4 Cell Viability Counter</td>
			<td style="width:157px;">Nexcelom&#160; Bioscience</td>
			<td style="width:156px;"></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...

Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

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 ...

The overall goal of these procedures is to quickly isolate highly purified neutrophils from human blood samples by density gradient centrifugation to investigate their role in lymphoma cell sensitivity to cancer therapy using two D and three D cell culture models. These methods can help answer key questions about the interactions between the innate immune system and cancer such as what role does a neutrophil mediated tumor cell responses play in cancer therapy. The main advantage of density gradient separation is the ability to isolate pure immune cell populations in a short period of time without exposing the cells to function altering stimuli.To isolate the primary leukemic cells and neutrophils, begin by adding 15 milliliters of EDTA treated blood from chronic lymphocytic leukemia patient samples into individual, sterile 50 milliliter tubes. Dilute the blood with 15 milliliters of RPMI. Then carefully and slowly add 15 milliliters of room temperature density gradient solution under the blood, taking care not to mix the layers.After separating the cells by centrifugation, four distinct phases will be visible. The platelets and plasma at the top, a white ring of mononuclear cells, the density gradient solution, and the granulocytes and erythrocytes at the bottom. Using a plastic pasture pipette, first transfer the white ring of primary leukemic cells in a new 50 milliliter tube.Wash the cells with up to 50 milliliters of PBS with calcium and magnesium. And re-suspend the palate in five milliliters of fresh PBS. Then, add 45 more milliliters of PBS to the cells, and spin down the cells again.After the second wash, re-suspend the palate in complete RPMI medium. And count the number of viable primary leukemic cells. Next, remove the platelet plasma and density gradient layers, and add 25 milliliter of PBS and 25 milliliters of Dextrin in sodium chloride to the granulocyte erythrocyte phase.Mix the tubes ten times by inversion. Then, let the cells settle for 30 minutes at room temperature. At the end of the equilibration, transfer the upper red blood cell poor neutrophil layer into a sterile, 50 milliliter tube and spin down the cells.Resuspend the palate in five milliliters of PBS and lyse the red blood cells with 45 milliliters of red cell lysis buffer. After 15 minutes in the dark at room temperature, spin down the cells again and wash the palate in 50 milliliters of PBS. At the end of the wash, re-suspend the cells in the complete RPMI medium and count the number of viable neutrophils.Dilute both groups of cells to a 300, 000 cell per 50 microliters of RPMI concentration and divide the samples between the appropriate number of five milliliter fax tubes. After centrifugation, resuspend the pellets in 50 microliters of PBS supplemented with FBS. To determine the purity of the isolated cell subsets, label the aliquant of primary leukemic cells with anti humanCD19 antibody conjugated to APC and the aliquant of the purified neutrophils with a mixture of anti human antibodies as listed in the table for 30 minutes in the dark at four degrees Celsius.At the end of the incubation, wash the cells in 500 microliters of FBS supplemented PBS and resuspend the palates in 200 microliters of fresh FBS PBS. Then, analyze the purity of the cell populations by flow cytometry, taking care to color compensate the samples. To co-culture the primary leukemic cells with the autologist neutrophils, see 200, 000 cells per milliliter of leukemic cells alone or with autologist neutrophils in a 24 well plate in complete RPMI medium.Mix the cells carefully. Then treat the cells with Brutton's Tyrosine Kinase Inhibitor for 24 hours at 37 degrees Celsius and five percent CO2. The next day, collect an aliquant of the cells in a five milliliter plastic fax tube.Then, spin down the cells and wash the pellets in one milliliter of FBS PBS. After the second centrifugation, label the cells aninexine five and propidium iodide according to the manufacturer's instructions for ten minutes at room temperature in the dark. Then, analyze the neutrophils and primary leukemic cells by flow cytometry using the illustrated gating strategy.To set up a three D co-culture, add 50, 000 RL lymphoma b-cells alone or mix with HL60 differentiated cells in sterile 15 milliliter tubes. Spin down the samples and resuspend the pellets in 300 microliters of basement membrane matrix, using a one millimeter pipette tip with the opening cut to two to three millimeters. Next, incubate 300 microliters of the cells in each well of a 24 well plate for 30 minutes at 37 degrees Celsius and five percent CO2.At the end of the incubation, add one milliliter of complete RPMI medium to each well and return the threeD cultures to the incubator for another seven days with medium changes every two days. On day five, add ten nanomolar of incristine to the cultures. After one week of culture, aspirate the medium and wash each well two times with one milliliter of ice cold PBS.Then, add three milliliters of ice cold PBS with EDTA to each well and scrape the bottom of the wells with a 200 microliter pipette tip to detach the gels. Shake the plate gently on ice for 30 minutes. Then, transfer the cell suspensions to individual sterile 15 milliliter tubes for gentle shaking on ice for another 30 minutes.Confirm the appearance of a homogenous cell suspension. Then, spin down the cells followed by a PBS wash and resuspend the pellets in fresh PBS with FBS. Finally, label the cells with the appropriate antibodies and viability dyes as just demonstrated and analyze the RL and HL60 differentiated cells by flow cytometry using the illustrated gating strategy.Analyzing all the events of the isolated cell populations by flow cytometry demonstrates that this separation method results in a highly pure single peak CD19 expressing primary leukemic cell population. And a greater than 90 percent pure neutrophil population positive for the leukocyte common antigens CD45, and the CD15 and CD16 antigens expressed by mature neutrophils. Gaiting on the primary leukemic cells from neutrophil co cultures demonstrates that the neutrophils exert a protective effect on cell survival against the tyrosine inhibitor ibrutinib with a significantly higher number of viable primary leukemic cells compared to the culture of primary leukemic cells alone.Differentiated HL60 cells are more stable in three D cultures than neutrophils. After induction of their granulocytic development, these cells are smaller than undifferentiated HL60 cells and express higher levels of CD11B and CD38. Differentiated HL60 cells also exhibit multi lobed nuclei.These cells exhibit a protective effect on the RL cells with a significantly higher number of viable RL cells in three D co cultures after vincrostin treatment compared to vincrostin treated lymphoma cells cultured alone. Once mastered, the density gradient centrifugation method can be completed in 90 minutes. After their development, these techniques paved the way for researchers in the field of cancer biology to explore the impact of various white blood cells on tumor malignancy in preclinical models and patient samples.After watching this video, you should have a good understanding of how to quickly and reliably isolate mononuclear cells and the neutrophils, analyze the effect of neutrophils on mediating tumor cell responses to anti cancer agents, and set up a three D co culture.

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

Research

JoVE Journal

Immunology and Infection

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...