Immuno-analysis of microparticles: probing at the limits of detection
Identifieur interne : 000F25 ( Pmc/Checkpoint ); précédent : 000F24; suivant : 000F26Immuno-analysis of microparticles: probing at the limits of detection
Auteurs : Sharissa L. Latham [Australie, Allemagne] ; Natalia Tiberti [Australie] ; Naveena Gokoolparsadh [Australie] ; Karen Holdaway [Australie] ; Pierre Olivier Couraud [France] ; Georges E. R. Grau [Australie] ; Valery Combes [Australie]Source :
- Scientific Reports [ 2045-2322 ] ; 2015.
Abstract
Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic
Url:
DOI: 10.1038/srep16314
PubMed: 26553743
PubMed Central: 4639787
Affiliations:
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Latham</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Vascular Immunology Unit, Sydney Medical School, The University of Sydney</institution>, NSW,<country>Australia</country></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Institute for Biophysical Chemistry, Hannover Medical School</institution>, Hannover,<country>Germany</country></nlm:aff><country xml:lang="fr">Allemagne</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Tiberti, Natalia" sort="Tiberti, Natalia" uniqKey="Tiberti N" first="Natalia" last="Tiberti">Natalia Tiberti</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Vascular Immunology Unit, Sydney Medical School, The University of Sydney</institution>, NSW,<country>Australia</country></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Gokoolparsadh, Naveena" sort="Gokoolparsadh, Naveena" uniqKey="Gokoolparsadh N" first="Naveena" last="Gokoolparsadh">Naveena Gokoolparsadh</name><affiliation wicri:level="1"><nlm:aff id="a3"><institution>Australian Centre for Microscopy and Microanalysis, The University of Sydney</institution>, NSW,<country>Australia</country></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Holdaway, Karen" sort="Holdaway, Karen" uniqKey="Holdaway K" first="Karen" last="Holdaway">Karen Holdaway</name><affiliation wicri:level="1"><nlm:aff id="a4"><institution>Beckman</institution> Coulter<country>Australia, 23-27 Chaplin Drive, Lane Cove West, NSW 2066, Australia</country></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>The University of Chicago Medicine, 5841 South Maryland Avenue, MC 2115 Chicago</wicri:regionArea><wicri:noRegion>MC 2115 Chicago</wicri:noRegion></affiliation></author><author><name sortKey="Olivier Couraud, Pierre" sort="Olivier Couraud, Pierre" uniqKey="Olivier Couraud P" first="Pierre" last="Olivier Couraud">Pierre Olivier Couraud</name><affiliation wicri:level="1"><nlm:aff id="a5"><institution>Inserm, U1016, Institut Cochin</institution>, Paris,<country>France</country></nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a6"><institution>Cnrs, UMR8104</institution>, Paris,<country>France</country></nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a7"><institution>Univ Paris Descartes</institution>, Paris,<country>France</country></nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Grau, Georges E R" sort="Grau, Georges E R" uniqKey="Grau G" first="Georges E. R." last="Grau">Georges E. R. Grau</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Vascular Immunology Unit, Sydney Medical School, The University of Sydney</institution>, NSW,<country>Australia</country></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Combes, Valery" sort="Combes, Valery" uniqKey="Combes V" first="Valery" last="Combes">Valery Combes</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Vascular Immunology Unit, Sydney Medical School, The University of Sydney</institution>, NSW,<country>Australia</country></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a8"><institution>Faculty of Science, School of Life Sciences, University of Technology Sydney</institution>, NSW,<country>Australia</country></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic <italic>in vitro</italic> system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ~60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30–40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Gonzalez Quintero, V H" uniqKey="Gonzalez Quintero V">V. H. Gonzalez-Quintero</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kumpers, P" uniqKey="Kumpers P">P. Kumpers</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Amabile, N" uniqKey="Amabile N">N. Amabile</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nozaki, T" uniqKey="Nozaki T">T. Nozaki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baron, M" uniqKey="Baron M">M. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Sci Rep</journal-id><journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type="epub">2045-2322</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26553743</article-id><article-id pub-id-type="pmc">4639787</article-id><article-id pub-id-type="pii">srep16314</article-id><article-id pub-id-type="doi">10.1038/srep16314</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Immuno-analysis of microparticles: probing at the limits of detection</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Latham</surname><given-names>Sharissa L.</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Tiberti</surname><given-names>Natalia</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Gokoolparsadh</surname><given-names>Naveena</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Holdaway</surname><given-names>Karen</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Olivier Couraud</surname><given-names>Pierre</given-names></name><xref ref-type="aff" rid="a5">5</xref><xref ref-type="aff" rid="a6">6</xref><xref ref-type="aff" rid="a7">7</xref></contrib><contrib contrib-type="author"><name><surname>Grau</surname><given-names>Georges E. R.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Combes</surname><given-names>Valery</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a8">8</xref></contrib><aff id="a1"><label>1</label><institution>Vascular Immunology Unit, Sydney Medical School, The University of Sydney</institution>, NSW,<country>Australia</country></aff><aff id="a2"><label>2</label><institution>Institute for Biophysical Chemistry, Hannover Medical School</institution>, Hannover,<country>Germany</country></aff><aff id="a3"><label>3</label><institution>Australian Centre for Microscopy and Microanalysis, The University of Sydney</institution>, NSW,<country>Australia</country></aff><aff id="a4"><label>4</label><institution>Beckman</institution> Coulter<country>Australia, 23-27 Chaplin Drive, Lane Cove West, NSW 2066, Australia</country></aff><aff id="a5"><label>5</label><institution>Inserm, U1016, Institut Cochin</institution>, Paris,<country>France</country></aff><aff id="a6"><label>6</label><institution>Cnrs, UMR8104</institution>, Paris,<country>France</country></aff><aff id="a7"><label>7</label><institution>Univ Paris Descartes</institution>, Paris,<country>France</country></aff><aff id="a8"><label>8</label><institution>Faculty of Science, School of Life Sciences, University of Technology Sydney</institution>, NSW,<country>Australia</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>valery.combes@uts.edu.au or valery.combes@sydney.edu.au</email></corresp></author-notes><pub-date pub-type="epub"><day>10</day><month>11</month><year>2015</year></pub-date><pub-date pub-type="collection"><year>2015</year></pub-date><volume>5</volume><elocation-id>16314</elocation-id><history><date date-type="received"><day>26</day><month>06</month><year>2015</year></date><date date-type="accepted"><day>07</day><month>10</month><year>2015</year></date></history><permissions><copyright-statement>Copyright © 2015, Macmillan Publishers Limited</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>Macmillan Publishers Limited</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract><p>Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic <italic>in vitro</italic> system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ~60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30–40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>Endothelial MP quantitation and characterisation by FCM.</title><p>Culture supernatants were collected from control or TNF activated (100 ng/ml overnight) hCMEC/D3 cells and labelled with either annexin V-FITC or lactaherin-FITC in combination with either CD54-PE or CD105-PE. (<bold>a</bold>) The total numbers of MP per 10<sup>3</sup> cells are shown with dark grey bars representing the levels of PS + MP, light grey bars representing double + MP and white bars indicating the numbers of Ag + MP. Annexin V is shown on the left hand side of each graph vs lactadherin on the right, CD54 is on top and CD105 below (mean ± SE; *p < 0.05, **p < 0.01, ***p < 0.0001). (<bold>b</bold>) The numbers of annexin V and lactadherin positive MP per 10<sup>3</sup> cells were compared between the 6 replicate wells analysed over 3 individual experiments (n = 18). Spearman’s test shows a significant correlation between the two PS labels (r = 0.96; p < 0.0001). (<bold>c</bold>) Mean fluorescence intensity (MFI) data for each individual label were pooled (n = 36) and differences between resting (NS) and TNF MP were assessed (mean ± SE; ***p < 0.0001). (<bold>d</bold>) Median fluorescence intensity for arbitrary size gates (<bold>a</bold>–<bold>e</bold>) (smallest to largest) for each individual label (mean ± SE; *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001).</p></caption><graphic xlink:href="srep16314-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>Quantitative EM shows no influence of vesiculation agonist on MP size.</title><p>(<bold>a</bold>) Representative transmission electron micrograph of purified, negatively stained MP collected with the JEOL 1400 TEM at 20 000 X Mag. (<bold>b</bold>) Minimum vs maximum Feret diameters (μm) for all MP analysed (n = 1690) with Image J software, illustrating overall endothelial MP size distribution. A significant correlation was confirmed between the two parameters (Spearman’s r = 0.96; p < 0.0001). (<bold>c</bold>) No significant differences in the distribution (Median, 25–75% percentile and range) of maximum Feret diameters (μm) between resting (NS) and TNF MP were observed (Mann Whitney test; p = 0.38).</p></caption><graphic xlink:href="srep16314-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>A direct correlation between MP size and marker expression.</title><p>(<bold>a</bold>) Immuno-gold labelled MP grids were imaged at 60 000X Mag with the JEOL 1400 TEM. All images are to the same scale where scale bars are measured in micron. Closed arrow-heads indicate gold positive MP whilst open arrow-heads indicate gold-negative MP. (<bold>b</bold>) Spearman’s correlation shows a significant relationship between the size of MP, measured as the maximum Feret diameter (μm), and the number of gold particles bound per MP for the annexin V label (r = 0.68; p < 0.0001). (<bold>c</bold>) The median size, measured as the maximum Feret diameter in μm, was compared between gold-positive and gold-negative MP for each of the markers of interest (Mann Whitney test; ***p < 0.0001).</p></caption><graphic xlink:href="srep16314-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>Comparative dual labelled FCM and immuno-EM show distinct differences in the percentage of Ag+ MP.</title><p>(<bold>a</bold>) Purified MP samples dual immuno-labelled with either annexin V or lactadherin (10 nm gold, closed arrowheads) followed by anti-CD54 or CD105 Abs (15 nm gold, open arrowheads) were negatively labelled and imaged at 100 000 X Mag on the JEOL 1400 TEM. (<bold>b,c</bold>) The percentages of PS+ (dark grey), double + (light grey) and Ag + (white) were determined from the total numbers of MP analysed for each marker combination and for both the FCM (n = 3) and immuno-EM (n = 1) experiments. CD54 data is shown on the top row whilst CD105 is below for both annexin V (<bold>b</bold>) and lactadherin (<bold>c</bold>) labels.</p></caption><graphic xlink:href="srep16314-f4"></graphic></fig><fig id="f5"><label>Figure 5</label><caption><title>Flow cytometry setup (methods) (<bold>a–d</bold>) Definition of the MP gate on FSC vs SSC cytograms.</title><p>(<bold>a</bold>) Fluorescent beads were used to set the protocol and allow evaluation of its stability. (<bold>b</bold>) PBS analysis demonstrates the purity of the machine where all the events detected correspond to the electronic noise of the instrument. (<bold>c</bold>) Analysis of a cell culture supernatant shows that the majority of the events fall with the MP gate. (<bold>d</bold>) Arbitrary gates (<bold>a–e</bold>) divide the MP gate to assess the relationship between MP size and Ag labelling. (<bold>e,f</bold>) examples of excluding swarm detection. (<bold>e</bold>) Number of MP positive for Annexin V detected in a serial dilution of the culture supernatant. (<bold>f</bold>) The time of Flight (TOF) parameter within a positive MP population where the left gate represent the single events and the right gate indicates the coincident events. (<bold>g,h</bold>) Examples of labelling combinations. CD105 vs annexin V and lactadherin on TNF-stimulated cells (<bold>g</bold>, left and right respectively). CD54 vs annexin V staining on resting vs TNF-stimulated cells (<bold>h</bold>, left and right respectively). MP were counted in the upper left quadrant for Ag only (Ag+), upper right for double labelled (double), lower right of PS marker only (PS+) and lower left for negative (Neg).</p></caption><graphic xlink:href="srep16314-f5"></graphic></fig><table-wrap position="float" id="t1"><label>Table 1</label><caption><title>The numbers and percentages of MP as assessed by FCM.</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center" char="±"></col><col align="center" char="±"></col><col align="center" char="±"></col><col align="center" char="±"></col></colgroup><thead valign="bottom"><tr><th rowspan="2" align="left" valign="top" charoff="50"> </th><th align="center" valign="top" char="±" charoff="50">NS</th><th align="center" valign="top" char="±" charoff="50">TNF</th><th align="center" valign="top" char="±" charoff="50">NS</th><th align="center" valign="top" char="±" charoff="50">TNF</th></tr><tr><th colspan="2" align="center" valign="top" char="±" charoff="50">Annexin V</th><th colspan="2" align="center" valign="top" char="±" charoff="50">Lactadherin</th></tr></thead><tbody valign="top"><tr><td colspan="5" align="left" valign="top" charoff="50">CD54 and PS dual labelled samples (mean MP number/10<sup>3</sup> cells ± SEM)</td></tr><tr><td align="left" valign="top" charoff="50"> CD54</td><td align="center" valign="top" char="±" charoff="50">3.7 ± 0.3</td><td align="center" valign="top" char="±" charoff="50">9.1 ± 0.8</td><td align="center" valign="top" char="±" charoff="50">3.2 ± 0.3</td><td align="center" valign="top" char="±" charoff="50">9.7 ± 1.5</td></tr><tr><td align="left" valign="top" charoff="50"> PS</td><td align="center" valign="top" char="±" charoff="50">129.8 ± 5.8</td><td align="center" valign="top" char="±" charoff="50">235.1 ± 2.6*</td><td align="center" valign="top" char="±" charoff="50">108 ± 5</td><td align="center" valign="top" char="±" charoff="50">186 ± 6.9*</td></tr><tr><td align="left" valign="top" charoff="50"> Double</td><td align="center" valign="top" char="±" charoff="50">78.9 ± 3.4</td><td align="center" valign="top" char="±" charoff="50">220.9 ± 14.4*</td><td align="center" valign="top" char="±" charoff="50">88.2 ± 3.3</td><td align="center" valign="top" char="±" charoff="50">239.2 ± 14.9*</td></tr><tr><td align="left" valign="top" charoff="50"> Total</td><td align="center" valign="top" char="±" charoff="50">212.5 ± 8.6</td><td align="center" valign="top" char="±" charoff="50">465.1 ± 21.2*</td><td align="center" valign="top" char="±" charoff="50">199.4 ± 7.4</td><td align="center" valign="top" char="±" charoff="50">435.4 ± 16.2*</td></tr><tr><td colspan="5" align="left" valign="top" charoff="50">CD105 and PS dual labelled samples (mean MP number/10<sup>3</sup> cells ± SEM)</td></tr><tr><td align="left" valign="top" charoff="50"> CD105</td><td align="center" valign="top" char="±" charoff="50">8.2 ± 0.5</td><td align="center" valign="top" char="±" charoff="50">8.3 ± 0.3</td><td align="center" valign="top" char="±" charoff="50">9.87 ± 1.8</td><td align="center" valign="top" char="±" charoff="50">22.5 ± 3.7<xref ref-type="fn" rid="t1-fn1">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> PS</td><td align="center" valign="top" char="±" charoff="50">158 ± 10.8</td><td align="center" valign="top" char="±" charoff="50">347.3 ± 9.8*</td><td align="center" valign="top" char="±" charoff="50">121.2 ± 5.8</td><td align="center" valign="top" char="±" charoff="50">250.3 ± 12.4<xref ref-type="fn" rid="t1-fn1">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> Double</td><td align="center" valign="top" char="±" charoff="50">61.5 ± 2.5</td><td align="center" valign="top" char="±" charoff="50">130.1 ± 7.4*</td><td align="center" valign="top" char="±" charoff="50">78.1 ± 3.6</td><td align="center" valign="top" char="±" charoff="50">185.1 ± 9.7<xref ref-type="fn" rid="t1-fn1">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> Total</td><td align="center" valign="top" char="±" charoff="50">231 ± 11.3</td><td align="center" valign="top" char="±" charoff="50">485.8 ± 3.7*</td><td align="center" valign="top" char="±" charoff="50">209.2 ± 8.9</td><td align="center" valign="top" char="±" charoff="50">457.9 ± 23.2<xref ref-type="fn" rid="t1-fn1">*</xref></td></tr><tr><td colspan="5" align="left" valign="top" charoff="50">Fluorescence positivity for individual labels (% positive (% range))</td></tr><tr><td align="left" valign="top" charoff="50"> PS</td><td align="center" valign="top" char="±" charoff="50">31 (24–44)</td><td align="center" valign="top" char="±" charoff="50">48 (41–62)</td><td align="center" valign="top" char="±" charoff="50">44 (8–69)</td><td align="center" valign="top" char="±" charoff="50">52 (21–72)</td></tr><tr><td align="left" valign="top" charoff="50"> CD54<xref ref-type="fn" rid="t1-fn2">#</xref></td><td align="center" valign="top" char="±" charoff="50">NS: 17 (5–34)</td><td align="center" valign="top" char="±" charoff="50"> </td><td align="center" valign="top" char="±" charoff="50">TNF: 25 (17–45)</td><td align="center" valign="top" char="±" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"> CD105<xref ref-type="fn" rid="t1-fn2">#</xref></td><td align="center" valign="top" char="±" charoff="50">NS: 13 (3–32)</td><td align="center" valign="top" char="±" charoff="50"> </td><td align="center" valign="top" char="±" charoff="50">TNF: 17 (9–33)</td><td align="center" valign="top" char="±" charoff="50"> </td></tr><tr><td colspan="5" align="left" valign="top" charoff="50">Fluorescence positivity for multiple labels (% positive ± SEM)</td></tr><tr><td align="left" valign="top" charoff="50"> CD54/PS</td><td align="center" valign="top" char="±" charoff="50">37 ± 0.8</td><td align="center" valign="top" char="±" charoff="50">47 ± 1.6</td><td align="center" valign="top" char="±" charoff="50">44 ± 0.9</td><td align="center" valign="top" char="±" charoff="50">54 ± 1.7</td></tr><tr><td align="left" valign="top" charoff="50"> CD105/PS</td><td align="center" valign="top" char="±" charoff="50">28.4 ± 1.5</td><td align="center" valign="top" char="±" charoff="50">27.1 ± 1.1</td><td align="center" valign="top" char="±" charoff="50">37.6 ± 1.2</td><td align="center" valign="top" char="±" charoff="50">37.1 ± 1.3</td></tr></tbody></table><table-wrap-foot><fn id="t1-fn1"><p><sup>*</sup>Significant difference between non-stimulated (NS) and TNF activated conditions, Mann-Whitney test, p < 0.05.</p></fn><fn id="t1-fn2"><p><sup>#</sup>Data collated from both Annexin V and Lactadherin labelled experiments.</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t2"><label>Table 2</label><caption><title>Effect of vesiculation agonist and probe on marker expression and detection.</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col></colgroup><thead valign="bottom"><tr><th rowspan="2" align="left" valign="top" charoff="50">Labelling</th><th colspan="2" align="center" valign="top" charoff="50">NS<hr></hr></th><th colspan="2" align="center" valign="top" charoff="50">TNF<hr></hr></th><th rowspan="2" align="center" valign="top" charoff="50">P Value*</th></tr><tr><th align="center" valign="top" charoff="50">n</th><th align="center" valign="top" charoff="50">Median # Gold (Range)</th><th align="center" valign="top" charoff="50">n</th><th align="center" valign="top" charoff="50">Median # Gold (Range)</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">Annexin V</td><td align="center" valign="top" charoff="50">175</td><td align="center" valign="top" charoff="50">2 (1–72)</td><td align="center" valign="top" charoff="50">184</td><td align="center" valign="top" charoff="50">4 (1–213)</td><td align="center" valign="top" charoff="50">0.01</td></tr><tr><td align="left" valign="top" charoff="50">Lactadherin</td><td align="center" valign="top" charoff="50">177</td><td align="center" valign="top" charoff="50">3 (1–172)</td><td align="center" valign="top" charoff="50">239</td><td align="center" valign="top" charoff="50">3 (1–53)</td><td align="center" valign="top" charoff="50">0.734</td></tr><tr><td align="left" valign="top" charoff="50">CD54</td><td align="center" valign="top" charoff="50">63</td><td align="center" valign="top" charoff="50">1 (1–12)</td><td align="center" valign="top" charoff="50">140</td><td align="center" valign="top" charoff="50">2 (1–103)</td><td align="center" valign="top" charoff="50"><0.0001</td></tr><tr><td align="left" valign="top" charoff="50">CD105</td><td align="center" valign="top" charoff="50">52</td><td align="center" valign="top" charoff="50">1 (1–26)</td><td align="center" valign="top" charoff="50">67</td><td align="center" valign="top" charoff="50">1 (1–19)</td><td align="center" valign="top" charoff="50">0.998</td></tr></tbody></table><table-wrap-foot><fn id="t2-fn1"><p>NS: non-stimulated; TNF: tumour necrosis factor (100 ng/ml).</p></fn><fn id="t2-fn2"><p>n = the number of MP enumerated per condition.</p></fn><fn id="t2-fn3"><p>Median # gold (range) = the median number and range of gold particles bound per MP.</p></fn><fn id="t2-fn4"><p>*Kolmogorov-Smirnov comparison of gold distributions.</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t3"><label>Table 3</label><caption><title>Small size distribution and low immunogold binding in the PS-/Ag+ population.</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col></colgroup><thead valign="bottom"><tr><th rowspan="2" align="left" valign="top" charoff="50">Labelling</th><th colspan="3" align="center" valign="top" charoff="50">NS<hr></hr></th><th colspan="3" align="center" valign="top" charoff="50">TNF<hr></hr></th></tr><tr><th align="center" valign="top" charoff="50">n</th><th align="center" valign="top" charoff="50">Maximum Feret (μm)</th><th align="center" valign="top" charoff="50"><xref ref-type="fn" rid="t3-fn1">#</xref>Gold Bound</th><th align="center" valign="top" charoff="50">n</th><th align="center" valign="top" charoff="50">Maximum Feret (μm)</th><th align="center" valign="top" charoff="50"><xref ref-type="fn" rid="t3-fn1">#</xref>Gold Bound</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">AV + CD54</td><td align="center" valign="top" charoff="50">2</td><td align="center" valign="top" charoff="50">0.2–0.212</td><td align="center" valign="top" charoff="50">1</td><td align="center" valign="top" charoff="50">11</td><td align="center" valign="top" charoff="50">0.115–0.506</td><td align="center" valign="top" charoff="50">1–4</td></tr><tr><td align="left" valign="top" charoff="50">AV + CD105</td><td align="center" valign="top" charoff="50">6</td><td align="center" valign="top" charoff="50">0.19–0.415</td><td align="center" valign="top" charoff="50">1–4</td><td align="center" valign="top" charoff="50">4</td><td align="center" valign="top" charoff="50">0.141–0.296</td><td align="center" valign="top" charoff="50">1–2</td></tr><tr><td align="left" valign="top" charoff="50">Lact + CD54</td><td align="center" valign="top" charoff="50">5</td><td align="center" valign="top" charoff="50">0.204–0.438</td><td align="center" valign="top" charoff="50">1–2</td><td align="center" valign="top" charoff="50">17</td><td align="center" valign="top" charoff="50">0.138–0.516</td><td align="center" valign="top" charoff="50">1–8</td></tr><tr><td align="left" valign="top" charoff="50">Lact + CD105</td><td align="center" valign="top" charoff="50">7</td><td align="center" valign="top" charoff="50">0.184–0.266</td><td align="center" valign="top" charoff="50">1–6</td><td align="center" valign="top" charoff="50">5</td><td align="center" valign="top" charoff="50">0.146–0.446</td><td align="center" valign="top" charoff="50">1</td></tr></tbody></table><table-wrap-foot><fn id="t3-fn1"><p>NS: non-stimulated; TNF: tumour necrosis factor (100 ng/ml).</p></fn><fn id="t3-fn2"><p>n = the number of double positive MP for each labelling combination.</p></fn><fn id="t3-fn3"><p><sup>#</sup>Gold Bound = the range of gold particles counted on MP in each labelling combination.</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t4"><label>Table 4</label><caption><title>Mean Equivalent Soluble Fluorochrome (MESF) values for FITC and PE in this high-sensitivity FCM system.</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center"></col><col align="center"></col><col align="center"></col></colgroup><thead valign="bottom"><tr><th colspan="2" align="left" valign="top" charoff="50">FITC<hr></hr></th><th colspan="2" align="center" valign="top" charoff="50">PE<hr></hr></th></tr><tr><th align="left" valign="top" charoff="50">Relative Channel</th><th align="center" valign="top" charoff="50">MESF</th><th align="center" valign="top" charoff="50">Relative Channel</th><th align="center" valign="top" charoff="50">MESF</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">0.49</td><td align="center" valign="top" charoff="50">954</td><td align="center" valign="top" charoff="50">0.32</td><td align="center" valign="top" charoff="50">621</td></tr><tr><td align="left" valign="top" charoff="50">1.12</td><td align="center" valign="top" charoff="50">2289</td><td align="center" valign="top" charoff="50">0.83</td><td align="center" valign="top" charoff="50">1734</td></tr><tr><td align="left" valign="top" charoff="50">2.29</td><td align="center" valign="top" charoff="50">6351</td><td align="center" valign="top" charoff="50">2.27</td><td align="center" valign="top" charoff="50">5044</td></tr><tr><td align="left" valign="top" charoff="50">7.74</td><td align="center" valign="top" charoff="50">16746</td><td align="center" valign="top" charoff="50">6.23</td><td align="center" valign="top" charoff="50">14029</td></tr><tr><td align="left" valign="top" charoff="50">21.87</td><td align="center" valign="top" charoff="50">49198</td><td align="center" valign="top" charoff="50">18.05</td><td align="center" valign="top" charoff="50">41359</td></tr><tr><td align="left" valign="top" charoff="50">64.33</td><td align="center" valign="top" charoff="50">146994</td><td align="center" valign="top" charoff="50">56.01</td><td align="center" valign="top" charoff="50">130228</td></tr><tr><td align="left" valign="top" charoff="50">127.96</td><td align="center" valign="top" charoff="50">281594</td><td align="center" valign="top" charoff="50">126.77</td><td align="center" valign="top" charoff="50">286384</td></tr></tbody></table></table-wrap></floats-group></pmc><affiliations><list><country><li>Allemagne</li><li>Australie</li><li>France</li></country></list><tree><country name="Australie"><noRegion><name sortKey="Latham, Sharissa L" sort="Latham, Sharissa L" uniqKey="Latham S" first="Sharissa L." last="Latham">Sharissa L. Latham</name></noRegion><name sortKey="Combes, Valery" sort="Combes, Valery" uniqKey="Combes V" first="Valery" last="Combes">Valery Combes</name><name sortKey="Combes, Valery" sort="Combes, Valery" uniqKey="Combes V" first="Valery" last="Combes">Valery Combes</name><name sortKey="Gokoolparsadh, Naveena" sort="Gokoolparsadh, Naveena" uniqKey="Gokoolparsadh N" first="Naveena" last="Gokoolparsadh">Naveena Gokoolparsadh</name><name sortKey="Grau, Georges E R" sort="Grau, Georges E R" uniqKey="Grau G" first="Georges E. R." last="Grau">Georges E. R. Grau</name><name sortKey="Holdaway, Karen" sort="Holdaway, Karen" uniqKey="Holdaway K" first="Karen" last="Holdaway">Karen Holdaway</name><name sortKey="Tiberti, Natalia" sort="Tiberti, Natalia" uniqKey="Tiberti N" first="Natalia" last="Tiberti">Natalia Tiberti</name></country><country name="Allemagne"><noRegion><name sortKey="Latham, Sharissa L" sort="Latham, Sharissa L" uniqKey="Latham S" first="Sharissa L." last="Latham">Sharissa L. Latham</name></noRegion></country><country name="France"><noRegion><name sortKey="Olivier Couraud, Pierre" sort="Olivier Couraud, Pierre" uniqKey="Olivier Couraud P" first="Pierre" last="Olivier Couraud">Pierre Olivier Couraud</name></noRegion><name sortKey="Olivier Couraud, Pierre" sort="Olivier Couraud, Pierre" uniqKey="Olivier Couraud P" first="Pierre" last="Olivier Couraud">Pierre Olivier Couraud</name><name sortKey="Olivier Couraud, Pierre" sort="Olivier Couraud, Pierre" uniqKey="Olivier Couraud P" first="Pierre" last="Olivier Couraud">Pierre Olivier Couraud</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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