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 ...

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