Name: quantitative imaging of lineage-specific toll-like receptor-mediated signaling in monocytes and dendritic cells from small samples of human blood
Uploaded: 2012-04-16T13:00:00
Description: Watch this Scientific Journal Video about Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood at JoVE.com

This work was supported in part by the NIH (N01 HHSN272201100019C, U19 AI 089992, and the NCRR/GCRC Program M01-RR00125). The authors declare no competing financial interests and thank Dr. Mark Shlomchik, director of the Yale Cell Sorter Core Facility.

1. Isolation and Stimulation of Immune Cells
<ol>
	<li>Obtain 10-50 ml heparinized blood from healthy volunteers after written informed consent under the guidelines of the local Institutional Review Board. For studies of aging or disease pathogenesis, exclusion and inclusion criteria for each volunteer or patient must be explicitly determined.</li>
	<li>Isolate human peripheral blood mononuclear cells (PBMCs) using CPT tubes or Ficoll-Paque Plus according to the manufacturer's instructions (GE Healthcare, NJ) and condu...

<ol>
	<li>Linton, P. J., Dorshkind, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+lymphocyte+development+and+function.">Age-related changes in lymphocyte development and function.</a> Nat. Immunol. 5, 133-139 (2004).</li><li>Shaw, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+Human+Toll-like+Receptor+Function+in+Aging.">Dysregulation of Human Toll-like Receptor Function in Aging.</a> Ageing Research Reviews. 10, 346-353 (2011).</li><li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+and+innate+immunity.">Toll-like receptors and innate immunity.</a> Nature Rev. Immunol. 1, 135-145 (2001).</li><li>van Duin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+Defect+in+Human+TLR1+Function+and+Expression.">Age-associated Defect in Human TLR1 Function and Expression.</a> J. Immunol. 178, 970-975 (2007).</li><li>Panda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+decrease+in+TLR+function+in+primary+human+dendritic+cells+predicts+influenza+vaccine+response.">Age-associated decrease in TLR function in primary human dendritic cells predicts influenza vaccine response.</a> J. Immunol. 184, 2518-2527 (2010).</li><li>Kong, K. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+TLR3+impairs+the+innate+immune+response+to+West+Nile+virus+in+the+elderly.">Dysregulation of TLR3 impairs the innate immune response to West Nile virus in the elderly.</a> J. Virol. 82, 7613-7623 (2008).</li><li>Qian, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+interferon+signaling+in+dendritic+cells+from+older+donors+infected+in+vitro+with+West+Nile+virus.">Impaired interferon signaling in dendritic cells from older donors infected in vitro with West Nile virus.</a> J. Infect. Dis. 203, 1415-1424 (2011).</li><li>Hayden, M. S., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB+in+immunobiology.">NF-kappaB in immunobiology.</a> Cell research. 21, 223-244 (2011).</li><li>Qian, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+elevation+in+TLR5+leads+to+increased+inflammatory+responses+in+the+elderly.">Age-associated elevation in TLR5 leads to increased inflammatory responses in the elderly.</a> Aging Cell. 11, 104-110 (2011).</li><li>George, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+nuclear+translocation+events+using+similarity+analysis+of+multispectral+cellular+images+obtained+in+flow.">Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow.</a> Journal of immunological. 311, 117-129 (2006).</li></ol>

The critical steps in use of ImageStream for translational studies are the selection of relevant comparison groups and optimization and validation of antibody specificity. Differences between subject groups in the labeled target will be readily apparent through histograms and cell images. In combination with a thorough analysis of changes in relevant receptors and signaling pathways, these data will provide valuable insight into mechanisms that underlie immune dysregulation in specialized cohorts. The technique will b...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Ficoll-Paque Plus</td>
 <td>GE Healthcare </td>
 <td>17-1440-03</td>
 </tr>
 <tr>
 <td>R848</td>
 <td>Invivogen</td>
 <td>tlrl-r848</td>
 </tr>
 <tr>
 <td>Paraformaldehyde (16%) </td>
 <td>Electron Microscopy Sciences </td>
 <td>15710</td>
 </tr>
 <tr>
 <td>BD Perm/Wash</td>
 <td>BD Biosciences</td>
 <td>554723</td>
 </tr>
 <tr>
 <td>CD14-PE</td>
 <td>eBioscience</td>
 <td>12-0149-41</td>
 </tr>
 <tr>
 <td>CD4-PacBlue</td>
 <td>BD Pharmingen</td>
 <td>68436</td>
 </tr>
 <tr>
 <td>Lineage cocktail 1 (lin 1)-FITC</td>
 <td>BD Biosciences</td>
 <td>340546</td>
 </tr>
 <tr>
 <td>CD11c-PE</td>
 <td>BD Biosciences</td>
 <td>347637</td>
 </tr>
 <tr>
 <td>CD123-PE</td>
 <td>BD Biosciences</td>
 <td>340545</td>
 </tr>
 <tr>
 <td>rabbit anti-NF-&kappa;B (p65) </td>
 <td>SantaCruz </td>
 <td>SC-372 </td>
 </tr>
 <tr>
 <td>Alexa647 F(ab')2 goat anti-rabbit IgG </td>
 <td>Invitrogen </td>
 <td>A21246</td>
 </tr>
 <tr>
 <td>DAPI </td>
 <td>Invitrogen </td>
 <td>D21490</td>
 </tr>
 <tr>
 <td>ImageStream X</td>
 <td>Amnis</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Table 1. Specific reagents and equipment.
<table border="1">
 <tbody>
 <tr>
 <td>Cell type</td>
 <td>FITC</td>
 <td>PE</td>
 <td>Alexa 647</td>
 <td>PacBlue</td>
 </tr>
 <tr>
 <td>Monocyte</td>
 <td>&nbsp;</td>
 <td>CD14</td>
 <td>NF-&kappa;B (p65)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>mDC</td>
 <td>Lin1</td>
 <td>CD11c</td>
 <td>NF-&kappa;B (p65)</td>
 <td>CD4</td>
 </tr>
 <tr>
 <td>pDC</td>
 <td>Lin1</td>
 <td>CD123</td>
 <td>NF-&kappa;B (p65)</td>
 <td>CD4</td>
 </tr>
 </tbody>
</table>
Table 2. Antibody panel for cell lineage staining of signaling pathways
Monocyte: CD14+ mDC: Lin1-, CD4dim, CD11c+ pDC: Lin1-, CD4dim, CD123+ Lin1 (lineage) marker includes CD3, CD14, CD19, CD20, CD56 markers for monocytes, NK cells, T &amp; B cells.

quantitative imaging of lineage-specific toll-like receptor-mediated signaling in monocytes and dendritic cells from small samples of human blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

Watch this Scientific Journal Video about Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood at JoVE.com

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Research

JoVE Journal

Immunology and Infection

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...