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.

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, ...

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

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 ...

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 ...

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 ...

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

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 ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

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 ...

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 ...

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

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 ...

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, ...