RSV A2 was kindly provided by Dr. R. de Swart (Erasmus MC, Rotterdam, The Netherlands). MV and GF are supported by the Virgo consortium, funded by the Dutch government project number FES0908, and by the Netherlands Genomics Initiative (NGI) project number 050-060-452. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Note:
Experiments with HRSV must be performed in a biological safety cabinet in a biosafety level 2 laboratory. Collect all viral waste and material that has been in contact with the virus for proper disposal due to biological hazards.
Special precaution must be taken when using potentially infected human blood.
It is crucial to use endotoxin-free reagents throughout all protocols.
1. Culturing of HRSV A2
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<ol>
	<li>Nair, H., Nokes, D. 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=Global+burden+of+acute+lower+respiratory+infections+due+to+respiratory+syncytial+virus+in+young+children:+a+systematic+review+and+meta-analysis.">Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis.</a> Lancet. 375, 1545-1555 (2010).</li><li>Falsey, A. R., Hennessey, P. A., Formica, M. A., Cox, C., Walsh, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+syncytial+virus+infection+in+elderly+and+high-risk+adults.">Respiratory syncytial virus infection in elderly and high-risk adults.</a> N. Eng. J. Med. , 352-1749 (2005).</li><li>Openshaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiviral+Immune+Responses+and+Lung+Inflammation+after+Respiratory+Syncytial+Virus+Infection.">Antiviral Immune Responses and Lung Inflammation after Respiratory Syncytial Virus Infection.</a> Proc. Am. Thorac. Soc. 2 (2), 121-125 (2005).</li><li>Openshaw, P. J., Tregoning, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+responses+and+disease+enhancement+during+respiratory+syncytial+virus+infection.">Immune responses and disease enhancement during respiratory syncytial virus infection.</a> Clin. Microbio. Rev. 18 (3), 541-555 (2005).</li><li>Villenave, R., Thavagnanam, 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=In+vitro+modeling+of+respiratory+syncytial+virus+infection+of+pediatric+bronchial+epithelium,+the+primary+target+of+infection+in+vivo.">In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 109 (13), 5040-5045 (2012).</li><li>Tulic, M. K., Hurrelbrink, R. J., Pr&#234;le, C. M., Laing, I. A., Upham, J. W., Le Souef, P., Sly, P. D., Holt, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR4+polymorphisms+mediate+impaired+responses+to+respiratory+syncytial+virus+and+lipopolysaccharide.">TLR4 polymorphisms mediate impaired responses to respiratory syncytial virus and lipopolysaccharide.</a> J. Immunol. 179 (1), 132-140 (2007).</li><li>Vissers, M., Remijn, 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=Respiratory+syncytial+virus+infection+augments+NOD2+signaling+in+an+IFN-&#946;-dependent+manner+in+human+primary+cells.">Respiratory syncytial virus infection augments NOD2 signaling in an IFN-&#946;-dependent manner in human primary cells.</a> Eur. J. Immunol. 42 (10), 2727-2735 (2012).</li><li>Cherukuri, A., Patton, K., Gasser, R. A., Zuo, F., Woo, J., Esser, M. T., Tang, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adults+65+years+old+and+older+have+reduced+number+of+functional+memory+T+cells+to+respiratory+syncytial+virus+fusion+protein.">Adults 65 years old and older have reduced number of functional memory T cells to respiratory syncytial virus fusion protein.</a> Clin. Vacc. Immunol. 20 (2), 239-247 (2013).</li><li>Shafique, M., Wilschut, J., de Haan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mucosal+and+systemic+immunity+against+respiratory+syncytial+virus+by+inactivated+virus+supplemented+with+TLR9+and+NOD2+ligands.">Induction of mucosal and systemic immunity against respiratory syncytial virus by inactivated virus supplemented with TLR9 and NOD2 ligands.</a> Vaccine. 30, 597-606 (2012).</li><li>Kwilas, S., Liesman, R. M., Zhang, L., Walsh, E., Pickles, R. J., Peeples, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+syncytial+virus+grown+in+Vero+cells+contains+a+truncated+attachment+protein+that+alters+its+infectivity+and+dependence+on+glycosaminoglycans.">Respiratory syncytial virus grown in Vero cells contains a truncated attachment protein that alters its infectivity and dependence on glycosaminoglycans.</a> J. Virol. 83 (20), 10710-10718 (2009).</li><li>Treuhaft, M. W., Beem, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+interfering+particles+of+respiratory+syncytial+virus.">Defective interfering particles of respiratory syncytial virus.</a> Infect. Immun. 37 (2), 439-444 (1982).</li><li>Korns Johnson, D., Homann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+and+improved+quantification+of+lymphocytic+choriomeningitis+virus+(LCMV)+titers+by+flow+cytometry.">Accelerated and improved quantification of lymphocytic choriomeningitis virus (LCMV) titers by flow cytometry.</a> PLoS One. 7 (5), e37337 (2012).</li><li>Wurfel, M. M., Park, W. Y., 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=Identification+of+high+and+low+responders+to+lipopolysaccharide+in+normal+subjects:+an+unbiased+approach+to+identify+modulators+of+innate+immunity.">Identification of high and low responders to lipopolysaccharide in normal subjects: an unbiased approach to identify modulators of innate immunity.</a> J. Immunol. 175 (4), 2570-2578 (2005).</li><li>Kimpen, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+syncytial+virus+and+asthma.+The+role+of+monocytes.">Respiratory syncytial virus and asthma. The role of monocytes.</a> Am. J. Respir. Crit. Care Med. 163, 7-9 (2001).</li><li>Wang, S. Z., Forsyth, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+neutrophils+with+respiratory+epithelial+cells+in+viral+infection.">The interaction of neutrophils with respiratory epithelial cells in viral infection.</a> Respirology. 5 (1), 1-10 (2000).</li><li>San-Juan-Vergara, H., Sampayo-Escobar, V., Reyes, N., Cha, B., Pacheco-Lugo, L., Wong, T., Peeples, M. E., Collins, P. L., Castano, M. E., Mohapatra, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol-rich+microdomains+as+docking+platforms+for+respiratory+syncytial+virus+in+normal+human+bronchial+epithelial+cells.">Cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells.</a> J. Virol. 86 (3), 1832-1843 (2012).</li><li>Davidson, D., Zaytseva, A., Miskolci, V., Castro-Alcaraz, S., Vancurova, I., Patel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profile+of+endotoxin-stimulated+leukocytes+of+term+new+born:+control+of+cytokine+gene+expression+by+interleukin-10.">Gene expression profile of endotoxin-stimulated leukocytes of term new born: control of cytokine gene expression by interleukin-10.</a> Plos One. 8 (1), e53641 (2013).</li></ol>

In this study, we demonstrate that infection of PBMC with HRSV is a fast and reliable model system in which inflammatory pathways can be studied. In order to stimulate PBMCs an HRSV stock has to be prepared and quantified.
Multiple cell lines are susceptible to HRSV infection. Cell lines most used for HRSV culturing are HEp-2, HeLa,&#160;and Vero cells. However, we would not recommend the usage of Vero cells for culturing of HRSV because it has been shown that culturing of HRSV in Vero cells r...

Human Respiratory Syncytial Virus (HRSV) is the most common cause of lower respiratory tract infections in children. Each year, over 33 million children under the age of five are infected with HRSV, leading to over three million hospitalizations and almost 200,000 deaths1. A growing body of evidence suggests that HRSV also poses a significant threat to the elderly and adults with underlying chronic illnesses2.
The majority of HRSV infections in young children presents with mild symptoms, comparable to the common cold, and do not require clinical intervention. However, a small proportion of patients require hospitalization and mechanical ventilation due to severe bronchiolitis.
Part of the pathogenesis of HRSV disease is the host&#39;s overexuberant and inadequate immune response to infection3,4. This is illustrated by several observations. The period of maximal illness is often preceded by the peak of viral infection and coincides better with cellular infiltration of infected tissues and the release of inflammatory cytokines3. Another line of evidence comes from the formalin inactivated HRSV (FI-RSV) trials in the 1960s. Instead of inducing protection in young infants, the vaccine resulted in an exaggerated immune response resulting in enhanced respiratory disease and higher morbidity and mortality.
Several models have been developed to study the pathogenesis of HRSV infections. Continuous cell lines, like HEp-2 and A549, have been used extensively to study HRSV infection in vitro. Epithelial cells are the primary targets of HRSV infection5, therefore a lot of focus has been on these cell types. However, the in vivo situation is much more complex and not limited to one cell type. In order to examine these complex interactions, several animal infection models have been developed to study HRSV pathogenesis. Thus far, two different strategies have been employed, focusing either on heterologous (nonhuman) models as well as cognate host-pneumovirus models. Examples of heterologous models for HRSV include chimpanzees, sheep, cotton rats and mice. RSV infection in its natural host has been studied in cattle and in mice, using bovine RSV and pneumovirus, respectively.
Whilst each of these models has provided important insights into disease pathogenesis, HRSV is highly adapted to humans. Therefore, as an addition to the cell lines and animal models currently used, we propose to use human primary PBMCs as a model for stimulation with HRSV. Human PBMCs can be obtained from a range of individuals to address specific research questions, including young children6, immunocompromised individuals as well as healthy adults7 or elderly individuals8. PBMCs can be used to study innate aspects of infection as well as the adaptive response to the virus. Activated inflammatory pathways important for the pathogenesis of HRSV disease can be studied at the mRNA and protein level. Further, viral infection of (subsets of) immune cells can be determined by flow cytometry. We have used this model previously to identify the synergistic effects of HRSV infection together with the bacterial ligand muramyl dipeptide (MDP) on the induction of proinflammatory cytokines7. We propose to use HRSV stimulation of human primary PBMCs as a robust, easy and fast in vitro model to study inflammatory pathways involved in the pathogenesis of HRSV disease.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
			<td>PBS without Ca2+ Mg2+ or phenol red</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14025-100</td>
			<td>HBSS, Calcium, Magnesium, no Phenol Red</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>31966-047</td>
			<td>DMEM, High Glucose, GlutaMAX, Pyruvate</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Invitrogen</td>
			<td>72400054</td>
			<td>RPMI 1640 Medium, GlutaMAX, HEPES</td>
		</tr>
		<tr>
			<td>Reduced Serum Medium (Opti-MEM)</td>
			<td>Invitrogen</td>
			<td>51985-026</td>
			<td>Opti-MEM I Reduced Serum Medium, GlutaMAX</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Greiner Bio-one</td>
			<td></td>
			<td>FBS (Fetal Bovine Serum)</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7030</td>
			<td>Albumin from bovine serum</td>
		</tr>
		<tr>
			<td>P/S</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>Penicillin-Streptomycin, liquid</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td>0.05% Trypsin-EDTA (1x), Phenol Red</td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903</td>
			<td>Sucrose BioXtra, &#8805;99.5%</td>
		</tr>
		<tr>
			<td>IgG2 anti-mouse-FITC</td>
			<td>BD Pharmingen</td>
			<td>555057</td>
			<td>Ms IgG2b K FITC isotype control</td>
		</tr>
		<tr>
			<td>Anti-NP-RSV-FITC</td>
			<td>Abcam</td>
			<td>ab25849</td>
			<td>Anti-Respiratory Syncytial Virus antibody [671] (FITC)</td>
		</tr>
		<tr>
			<td>Density gradient medium (Lymphoprep)</td>
			<td>Axis-Shield</td>
			<td>1114545</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA tubes</td>
			<td>BD Biosciences</td>
			<td>367525</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Merck Millipore</td>
			<td>1000141000</td>
			<td>Acetone for analysis</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material </td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td>Thinwall, Polyallomer, 38.5 ml, 25 mm x 89 mm</td>
		</tr>
		<tr>
			<td>SureSpin 630 Rotor with 36 ml buckets</td>
			<td>Sorvall</td>
			<td>79368</td>
			<td>Swinging bucket titanium rotor</td>
		</tr>
		<tr>
			<td>Sorvall WX80 Ultracentrifuge</td>
			<td>Sorvall</td>
			<td>46900</td>
			<td></td>
		</tr>
		<tr>
			<td>CB 150l CO2 Incubator</td>
			<td>Binder</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro model to study immune responses of human peripheral blood mononuclear cells to human respiratory syncytial virus infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

The HRSV A2 cultivation and purification method described here will give a yield between 2 x&#160;107 and 2 x&#160;108 FITC-50 quantified infectious viral particles/ml. Results of microscopic analysis, where FITC-stained infected cells were counted (results not shown), were compared to results of quantification by FACS (Figure 1). Both methods resulted in similar titers, although the FACS method had a much lower standard deviation between different experiments and was less laborious...

Watch this Scientific Journal Video about An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection at JoVE.com

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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Immunology and Infection

7.8K 次观看.  Radboud university medical center. Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

视频: An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and...

an in vitro co-infection model to study plasmodium falciparum-hiv-1 interactions in human primary monocyte-derived immune cells

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

isolation of tonsillar mononuclear cells to study ex vivo innate immune responses in a human mucosal lymphoid tissue

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal...

an in vitro model for measuring immune responses to malaria in the context of hiv co-infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

cryopreservation and bioenergetic evaluation of human peripheral blood mononuclear cells

Author Spotlight: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place...

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

xenograft skin model to manipulate human immune responses in vivo

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

generation and expansion of human cardiomyocytes from patient peripheral blood mononuclear cells

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear...

isolation of peripheral blood mononuclear cells from human buffy coats

In this video, we demonstrate the isolation of peripheral blood mononuclear cells, PBMCs, containing monocytes and lymphocytes from a human buffy coat by density gradient centrifugation. The isolated PBMCs can be used for further...

Isolation of Peripheral Blood Mononuclear Cells from Human Buffy Coats

PBMC Collection and Cryostorage for Mitochondrial Bioenergetics Analysis

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culturing of activated t cells from human peripheral blood mononuclear cells

In this video, we describe a protocol for human primary T cell activation. The incubation of na&#239;ve T cells with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, followed by interleukin-2 or interleukin-15 treatment, leads to the activation of na&#239;ve T cells and their differentiation and proliferation into effector or memory T...

Culturing of Activated T Cells from Human Peripheral Blood Mononuclear Cells

periodic acid-schiff staining of cells: an in vitro technique to detect glycogen levels in peripheral blood mononuclear cells

This video demonstrates the periodic acid-Schiff staining technique for assessing glycogen levels of peripheral blood mononuclear cells in vitro. The stain imparts a magenta color to intracellular glycogen granules, rendering them visible under a...

Periodic Acid-Schiff Staining of Cells: An In Vitro Technique to Detect Glycogen Levels in Peripheral Blood Mononuclear Cells

Add serum-free RPMI medium to the cells to a final volume of 50 milliliters, and gently invert the tube a few times before centrifuging. Then, resuspend the cells in 20 milliliters of serum-free medium for...

Next, add 12.5 microliters of beads per million cells at a 1 to 2 beads to cells ratio, and divide the cells into two conditions with around 5 million cells in each. Then, add the correct volume of cytokines to each condition, and transfer the cells to multi-well plates. Incubate the cells for three days at 37 degrees Celsius and 5% carbon...

a flow cytometry assay to identify immune cell subsets in peripheral blood mononuclear cells

This video demonstrates a flow cytometry-based technique using two fluorochromes to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, natural killer (NK) cells, and monocytes in human peripheral blood mononuclear cells (PBMCs). Upon incubating the cells with fluorochrome-conjugated antibodies against the surface markers, the cell subsets are identified using an optimal flow cytometric gating...

A Flow Cytometry Assay to Identify Immune Cell Subsets in Peripheral Blood Mononuclear Cells

Subsequently, allow the plate to sit for 10 minutes at room temperature for labeling of dead cells. For each sample, prepare 30 microliters of a mix containing all seven antibodies. At this stage, titrated antibodies against different target molecules and in different fluorochromes can be added as well. Centrifuge the 96-well plate at 350 times g for three minutes at room temperature, and carefully aspirate the supernatant without disturbing the cell...

contact-free co-culture model for the study of innate immune cell activation during respiratory virus infection

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral...

an in vitro model to study heterogeneity of human macrophage differentiation and polarization

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

an assay to measure the neutralizing antibody titer specific for respiratory syncytial virus

This video demonstrates an assay for measuring neutralizing antibodies against respiratory syncytial virus (RSV). Pre-incubation with serum containing RSV-neutralizing antibodies neutralizes the virus particles, reducing cellular infection and plaque formation. Virus-infected cells are then subjected to fixation, blocking, and fluorophore-conjugated antibody labeling for plaque visualization. This process enables the identification of the highest serum dilution that neutralizes fifty percent of...

An Assay to Measure the Neutralizing Antibody Titer Specific for Respiratory Syncytial Virus

assessing respiratory immune responses to haemophilus influenzae

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Next, add 100 microliters per well of the virus-serum mixture to the corresponding wells on the A549 cell plate according to the plate template provided in the manuscript, and incubate the plate at 37 degrees Celsius in 5% carbon dioxide for one hour. At the end of the incubation, use a pipette to remove 90 microliters per well, and add 100 microliters of the media containing 1X M 199, 1.5% CMC, and 2% FCS and penicillin-streptomycin mixture. Incubate the plate at 37 degrees Celsius in 5%...

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...