Name: Realtime, high-throughput microscopische kwantificering van de afgifte van extracellulaire vallen van menselijke neutrofielen en beoordeling van de farmacologie van antagonisten (Video) | JoVE Journal
Uploaded: 2024-10-18T14:20:00.000+00:00
Duration: 11 min 32 s
Description: Leer een real-time microscopiemethode voor kwantificering van NET-afgifte door menselijke neutrofielen. Deze techniek maakt high-throughput analyse van NETosis en farmacologische evaluatie van antagonisten mogelijk.

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<meta charset="utf8"></head><body>Real-time microscopie voor het bestuderen van NETosis-kinetiek en screeningremmers

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<li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+peptidylarginine+deiminase+4+%28PAD4%29+inhibitor+BMS-P5+blocks+formation+of+neutrophil+extracellular+traps+and+delays+progression+of+multiple+myeloma%5BTitle%5D)+AND+%22Mol+Cancer+Ther%22%5BJournal%5D)">A novel peptidylarginine deiminase 4 (PAD4) inhibitor BMS-P5 blocks formation of neutrophil extracellular traps and delays progression of multiple myeloma.</a> Mol Cancer Ther. 19 (7), 1530-1538 (2020).</li>
<li>Chirivi, R. G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-citrullinated+protein+antibodies+as+novel+therapeutic+drugs+in+rheumatoid+arthritis%5BTitle%5D)+AND+%22J+Clin+Cell+Immunol%22%5BJournal%5D)">Anti-citrullinated protein antibodies as novel therapeutic drugs in rheumatoid arthritis.</a> J Clin Cell Immunol. S6, 006(2013).</li>
<li>Van der Linden, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-citrullinated+histone+monoclonal+antibody+CIT-013%2C+a+dual+action+therapeutic+for+neutrophil+extracellular+trap-associated+autoimmune+diseases%5BTitle%5D)+AND+%22MAbs%22%5BJournal%5D)">Anti-citrullinated histone monoclonal antibody CIT-013, a dual action therapeutic for neutrophil extracellular trap-associated autoimmune diseases.</a> MAbs. 15 (1), 2281763(2023).</li>
<li>Chirivi, R. G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapeutic+ACPA+inhibits+NET+formation%3A+a+potential+therapy+for+neutrophil-mediated+inflammatory+diseases%5BTitle%5D)+AND+%22Cell+Mol+Immunol%22%5BJournal%5D)">Therapeutic ACPA inhibits NET formation: a potential therapy for neutrophil-mediated inflammatory diseases.</a> Cell Mol Immunol. 18 (6), 1528-1544 (2021).</li>
<li>Van der Linden, M., Westerlaken, G. H. A., Van Der Vlist, M., Van Montfrans, J., Meyaard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+signalling+and+kinetics+of+neutrophil+extracellular+trap+release+revealed+by+quantitative+live+imaging%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Differential signalling and kinetics of neutrophil extracellular trap release revealed by quantitative live imaging.</a> Sci Rep. 7 (1), 6529(2017).</li>
<li>Hoffmann, J. H. O., Schaekel, K., Gaiser, M. R., Enk, A. H., Hadaschik, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interindividual+variation+of+NETosis+in+healthy+donors%3A+introduction+and+application+of+a+refined+method+for+extracellular+trap+quantification%5BTitle%5D)+AND+%22Exp+Dermatol%22%5BJournal%5D)">Interindividual variation of NETosis in healthy donors: introduction and application of a refined method for extracellular trap quantification.</a> Exp Dermatol. 25 (11), 895-900 (2016).</li>
<li>Silva, L. M., Moutsopoulos, N., Bugge, T. H., Doyle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+imaging+and+quantification+of+neutrophil+extracellular+trap+formation%5BTitle%5D)+AND+%22Curr+Protoc%22%5BJournal%5D)">Live imaging and quantification of neutrophil extracellular trap formation.</a> Curr Protoc. 1 (7), e157(2021).</li>
<li>De Bont, C. M., Koopman, W. J. H., Boelens, W. C., Pruijn, G. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stimulus-dependent+chromatin+dynamics%2C+citrullination%2C+calcium+signalling+and+ROS+production+during+NET+formation%5BTitle%5D)+AND+%22Biochim+Biophys+Acta+Mol+Cell+Res%22%5BJournal%5D)">Stimulus-dependent chromatin dynamics, citrullination, calcium signalling and ROS production during NET formation.</a> Biochim Biophys Acta Mol Cell Res. 1865 (11 Pt A), 1621-1629 (2018).</li>
<li>Gajendran, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alleviation+of+arthritis+through+prevention+of+neutrophil+extracellular+traps+by+an+orally+available+inhibitor+of+protein+arginine+deiminase+4%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Alleviation of arthritis through prevention of neutrophil extracellular traps by an orally available inhibitor of protein arginine deiminase 4.</a> Sci Rep. 13 (1), 3189(2023).</li>
<li>Biron, B. M., Chung, C. -S., O'Brien, X. M., Chen, Y., Reichner, J. S., Ayala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cl-Amidine+prevents+histone+3+citrullination+and+neutrophil+extracellular+trap+formation%2C+and+improves+survival+in+a+murine+sepsis+model%5BTitle%5D)+AND+%22J+Innate+Immun%22%5BJournal%5D)">Cl-Amidine prevents histone 3 citrullination and neutrophil extracellular trap formation, and improves survival in a murine sepsis model.</a> J Innate Immun. 9 (1), 22-32 (2017).</li>
<li>Perdomo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+activation+and+NETosis+are+the+major+drivers+of+thrombosis+in+heparin-induced+thrombocytopenia%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Neutrophil activation and NETosis are the major drivers of thrombosis in heparin-induced thrombocytopenia.</a> Nat Commun. 10 (1), 1322(2019).</li>
<li>Lood, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps+enriched+in+oxidized+mitochondrial+DNA+are+interferogenic+and+contribute+to+lupus-like+disease%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease.</a> Nat Med. 22 (2), 146-153 (2016).</li>
<li>Kraaij, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+method+for+high-throughput+detection+and+quantification+of+neutrophil+extracellular+traps+reveals+ROS-independent+NET+release+with+immune+complexes%5BTitle%5D)+AND+%22Autoimmun+Rev%22%5BJournal%5D)">A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes.</a> Autoimmun Rev. 15 (6), 577-584 (2016).</li>
<li>De Buhr, N., Von Köckritz-Blickwede, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+neutrophil+extracellular+traps+become+visible%5BTitle%5D)+AND+%22J+Immunol+Res%22%5BJournal%5D)">How neutrophil extracellular traps become visible.</a> J Immunol Res. 2016, 1-13 (2016).</li>
<li>Brinkmann, V., Laube, B., Abu Abed, U., Goosmann, C., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps%3A+How+to+generate+and+visualize+them%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Neutrophil extracellular traps: How to generate and visualize them.</a> J Vis Exp. (36), e1724(2010).</li>
<li>Zhao, W., Fogg, D. K., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+image-based+quantitative+method+for+the+characterization+of+NETosis%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">A novel image-based quantitative method for the characterization of NETosis.</a> J Immunol Methods. 423, 104-110 (2015).</li>
<li>Gavillet, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometric+assay+for+direct+quantification+of+neutrophil+extracellular+traps+in+blood+samples%5BTitle%5D)+AND+%22Am+J+Hematol%22%5BJournal%5D)">Flow cytometric assay for direct quantification of neutrophil extracellular traps in blood samples.</a> Am J Hematol. 90 (12), 1155-1158 (2015).</li>
<li>Sil, P., Yoo, D., Floyd, M., Gingerich, A., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+throughput+measurement+of+extracellular+DNA+release+and+quantitative+NET+formation+in+human+neutrophils+in+vitro%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">High throughput measurement of extracellular DNA release and quantitative NET formation in human neutrophils in vitro.</a> J Vis Exp. (112), e52779(2016).</li>
<li>Gupta, S., Chan, D. W., Zaal, K. J., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput+real-time+imaging+technique+to+quantify+NETosis+and+distinguish+mechanisms+of+cell+death+in+human+neutrophils%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">A high-throughput real-time imaging technique to quantify NETosis and distinguish mechanisms of cell death in human neutrophils.</a> J Immunol. 200 (2), 869-879 (2018).</li>
<li>D'Cruz, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pseudokinase+MLKL+activates+PAD4-dependent+NET+formation+in+necroptotic+neutrophils%5BTitle%5D)+AND+%22Sci+Signal%22%5BJournal%5D)">The pseudokinase MLKL activates PAD4-dependent NET formation in necroptotic neutrophils.</a> Sci Signal. 11 (546), eaao1716(2018).</li>
<li>Nash, G., Abbitt, K., Tate, K., Jetha, K., Egginton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+the+mechanical+and+adhesive+behaviour+of+human+neutrophils+on+cooling+in+vitro%5BTitle%5D)+AND+%22Pflugers+Arch%22%5BJournal%5D)">Changes in the mechanical and adhesive behaviour of human neutrophils on cooling in vitro.</a> Pflugers Arch. 442 (5), 762-770 (2001).</li>
<li>Hundhammer, T., Gruber, M., Wittmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paralytic+impact+of+centrifugation+on+human+neutrophils%5BTitle%5D)+AND+%22Biomedicines%22%5BJournal%5D)">Paralytic impact of centrifugation on human neutrophils.</a> Biomedicines. 10 (11), 2896(2022).</li>
<li>Krémer, V., Godon, O., Bruhns, P., Jönsson, F., De Chaisemartin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+methods+determine+human+neutrophil+responses+after+stimulation%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Isolation methods determine human neutrophil responses after stimulation.</a> Front Immunol. 14, 1301183(2023).</li>
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</ol>

<table><tbody><tr><td>A-23187 Free Acid (Calcimycin)</td><td>Thermo Fisher</td><td>A1493</td><td></td></tr><tr><td>Ammonium chloride (NH4Cl)</td><td>Sigma Aldrich</td><td>A9434</td><td></td></tr><tr><td>Anti-Hen egg lysozyme (control antibody; cIgG)</td><td>CrownBio</td><td>C0001</td><td></td></tr><tr><td>APC-Cy7-conjugated mouse anti-human CD45 antibody (Clone 2D1)</td><td>Biolegend</td><td>368515</td><td>Gebruik bij 1 &micro;g/mL eindconcentratie</td></tr><tr><td>BD FACSCantoTM II system + FACSDiva software (versie 8.0.1)&nbsp;</td><td>BD</td><td>n/a</td><td>Flow cytometry systeem</td></tr><tr><td>Bovine Serum Albumin (BSA) Fraction V</td><td>Roche</td><td>10735108001</td><td></td></tr><tr><td>CaCl2 (1 M)</td><td>VWR</td><td>E506</td><td></td></tr><tr><td>Calcium pyrophosphate disposition disease (CPPD) crystals</td><td>InvivoGen</td><td>lrl-cppd</td><td></td></tr><tr><td>Cellometer Auto T4 Bright Field Cell Counter + analysis software (versie 3.3.9.5)</td><td>Nexcelom Bioscience</td><td>n/a</td><td>Bright field cell counter</td></tr><tr><td>CIT-013</td><td>Citryll B.V.</td><td>n/a</td><td></td></tr><tr><td>Costar black 96-well plate, clear bottom&nbsp;</td><td>Corning</td><td>3603</td><td>96-well plate #1</td></tr><tr><td>Dextran T500</td><td>Pharmacosmos</td><td>551005009006</td><td></td></tr><tr><td>DPBS (1x)</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Serena</td><td>S-FBS-SA-015</td><td></td></tr><tr><td>Ficoll&nbsp;</td><td>GE Healthcare</td><td>17-1440-02</td><td>Density gradient oplossing</td></tr><tr><td>FITC-conjugated mouse anti-human CD66b antibody (Clone G10F5)</td><td>eBioscience</td><td>17-0666-42</td><td>Gebruik bij 1,5 &micro;g/mL eindconcentratie</td></tr><tr><td>Fixable viability dye eFluor 506</td><td>eBioscience</td><td>65-0866-14</td><td></td></tr><tr><td>Flow cytometry analysis software</td><td>FLowJo</td><td>Versie 10.8.0</td><td></td></tr><tr><td>fMLP</td><td>Sigma Aldrich</td><td>47729-10MG-F</td><td></td></tr><tr><td>HEPES (1M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Human serum albumin (HSA)</td><td>VWR</td><td>31020</td><td></td></tr><tr><td>Human TruStain FcX&nbsp;</td><td>Biolegend</td><td>422302</td><td>Fc receptor blokker</td></tr><tr><td>IBIDI 96-well plate, clear bottom&nbsp;</td><td>IBIDI</td><td>89626</td><td>96-well plate #2</td></tr><tr><td>IncuCyte SX3 + analysis software (versie 2022B Rev1)&nbsp;</td><td>Satorius</td><td>n/a</td><td>Live cell microscopische analyse systeem</td></tr><tr><td>Monosodium urate crystals</td><td>InvivoGen</td><td>tlrl-msu</td><td></td></tr><tr><td>Mouse anti-histone H3 (citrulline R2 + R8 + R17) antibody&nbsp;</td><td>Cayman</td><td>17939</td><td>Clone 11D3; aangeduid als #1</td></tr><tr><td>Mouse anti-histone H4 (K8Ac + K12Ac + K16Ac) antibody (Clone KM-2)</td><td>Absolute Antibodies</td><td>Ab01681-2.0</td><td></td></tr><tr><td>Na2EDTA</td><td>Sigma Aldrich</td><td>E5134</td><td></td></tr><tr><td>NaCl (0.9%)</td><td>B. Braun</td><td>25900</td><td></td></tr><tr><td>Penicillin (5000 U/mL) - Streptomycin (5000 &micro;g/mL)</td><td>Gibco</td><td>15070-063</td><td></td></tr><tr><td>PerCP-Cy5.5-conjugated mouse anti-human CD16 antibody (Clone 3G8)</td><td>Biolegend</td><td>302027</td><td>Gebruik bij 0,33 &micro;g/mL eindconcentratie</td></tr><tr><td>PMA</td><td>Sigma Aldrich</td><td>P1585</td><td></td></tr><tr><td>Polyclonal rabbit anti-HSA antibody</td><td>Sigma Aldrich</td><td>A0433-2ml</td><td></td></tr><tr><td>Poly-L-Lysine (0.01%)</td><td>Sigma Aldrich</td><td>P4832</td><td></td></tr><tr><td>Potassium bicarbonate (KHCO3)</td><td>Sigma Aldrich</td><td>237205</td><td></td></tr><tr><td>Rabbit anti-histone H3 (citrulline R2 + R8 + R17) antibody&nbsp;</td><td>Abcam</td><td>ab281584</td><td>Multiclonal; aangeduid als #2</td></tr><tr><td>RPMI 1640 with GlutaMAXTM supplement&nbsp;</td><td>Gibco</td><td>61870-010</td><td>Cultuurmedium met L-glutamine</td></tr><tr><td>RPMI 1640 without phenol red&nbsp;</td><td>Gibco</td><td>11835-030</td><td>Cultuurmedium zonder fenolrood</td></tr><tr><td>Sodium azide (NaN3)</td><td>Sigma Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sterile H2O</td><td>Gibco</td><td>15230204</td><td></td></tr><tr><td>Sytox Green Nucleid Acid Stain&nbsp;</td><td>Thermo Fisher</td><td>S7020</td><td>DNA kleurstof</td></tr><tr><td>Trypan blue solution (0.4%)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Tween-20</td><td>Sigma Aldrich</td><td>P1379</td><td></td></tr><tr><td>Vacutainer blood tubes Li-Heparin (17 IU/mL)</td><td>BD</td><td>367526</td><td></td></tr></tbody></table>

realtime, high-throughput microscopische kwantificering van de afgifte van extracellulaire vallen van menselijke neutrofielen en beoordeling van de farmacologie van antagonisten

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists

Realtime, high-throughput microscopische kwantificering van de afgifte van extracellulaire vallen van menselijke neutrofielen en beoordeling van de farmacologie van antagonisten

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in ...

real-time-high-throughput-microscopic-quantification-human-neutrophil

Research

JoVE Journal

Biology

Biologie

Er worden high throughput-assays gepresenteerd die in combinatie uitstekende tools bieden om NET-afgifte van humane neutrofielen te kwantificeren.

High Throughput Meting van extracellulaire DNA los en Kwantitatieve NET Formation in menselijke neutrofielen<em&gt; In Vitro</em

Dit protocol beschrijft een zeer gevoelige en hoge doorvoer neutrofiele extracellulaire val (NET) assay voor de semi-automatische kwantificering van ex vivo NET vorming door immunofluorescentie driedimensionale confocale microscopie. Dit protocol kan worden gebruikt voor het evalueren van de netto vorming en afbraak na verschillende prikkels en kan worden gebruikt voor het bestuderen van potentiële NET-gerichte therapieën.

Een High-throughput Assay voor het beoordelen en kwantificeren van neutrofiele extracellulaire Trap vorming

We presenteren een geautomatiseerde high-throughput methode om neutrofiele extracellulaire vallen (NET's) te kwantificeren met behulp van het live cell analysesysteem, gekoppeld aan een membraanpermeabiliteit-afhankelijke dual-dye benadering.

Real-time High Throughput-techniek om de vorming van extracellulaire neutrofielen in menselijke neutrofielen te kwantificeren

Kwantificering van DNA dubbel-strengs strepen met behulp van γH2AX vorming als een moleculaire marker is uitgegroeid tot een onmisbaar instrument in straling biologie. Hier laten we zien het gebruik van een immunofluorescentie assay voor de kwantificering van γH2AX foci na blootstelling van cellen aan straling.

Kwantificering van γH2AX Foci in reactie op ioniserende straling

We beschrijven een methode voor de kwantitatieve, real-time meting van DNA glycosylase en AP endonuclease activiteiten in cel nucleaire lysaten. De test levert de tarieven van de DNA-reparatie-activiteit vatbaar zijn voor kinetische analyse en is aanpasbaar voor de kwantificering van DNA Repair activiteit in weefsel en tumor lysaten of met gezuiverde eiwitten.

Kwantitatieve, real-time analyse van Base excisie reparatie Activiteit in Cellysaten gebruik te maken van laesie-specifieke moleculaire Beacons

We stellen robuuste biochemische en microscopische methoden voor het...

Biochemische en High Throughput microscopische beoordeling van Fat Mass in<em&gt; Caenorhabditis Elegans</em

Zoogdierlijke cellen die het bacteriële bioluminescentie gencassette...

Autonoom Bioluminescente zoogdiercellen voor continue en real-time monitoring van de cytotoxiciteit

Surface Plasmon Resonance (SPR) is een label vrij werkwijze voor het detecteren van biomoleculaire interacties in real time. Hierin, een protocol voor een membraaneiwit: wordt receptor interactie experiment beschreven, terwijl de discussie over de voors en tegens van de techniek.

Real Time Metingen van membraaneiwit: Receptor Interacties gebruik Surface Plasmon Resonance (SPR)

Hier beschrijven we een celgebaseerde reportergen-test als waardevol hulpmiddel om chemische bibliotheken te screenen op verbindingen die post-transcriptionele controlemechanismen moduleren die worden uitgeoefend via 3’ UTR.

High-throughput screening voor Chemische Modulators van Post-transcriptioneel gereguleerde genen

Circulerende microRNAs zijn onlangs naar voren gekomen als veelbelovend en nieuwe biomarkers voor verschillende vormen van kanker en andere ziekten. Het doel van dit artikel is om drie verschillende probes gebaseerde real-time PCR-platforms en methoden die beschikbaar zijn om te kwantificeren en te bepalen de overvloed aan circulerende microRNAs zijn te bespreken.

Probe-gebaseerde real-time PCR Benaderingen voor kwantitatieve meting van microRNAs

Leer een real-time microscopiemethode voor kwantificering van NET-afgifte door menselijke neutrofielen. Deze techniek maakt high-throughput analyse van NETosis en farmacologische evaluatie van antagonisten mogelijk.

Realtime, high-throughput microscopische kwantificering van de afgifte van extracellulaire vallen van menselijke neutrofielen en beoordeling van de farmacologie van antagonisten (Video) | JoVE Journal

Realtime, high-throughput microscopische kwantificering van de afgifte van extracellulaire vallen van menselijke neutrofielen en beoordeling van de farmacologie van antagonisten

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