Bu çalışma, Avusturya Bilim Fonu&#39;nun (FWF) hibe P18820-B13 tarafından finanse edildi. Etik bir bildirimde: Çalışma, 1983 yılında gözden geçirilmiş ve katılan tüm hastaların yazılı bilgilendirilmiş onam verdi Helsinki Declaraction uygun Salzburg Üniversitesi, İnsanlar ve / veya Hayvanlar katılımı Deneyler (PLUS_Ethik_090514) Etik Komitesi tarafından kabul edildi.

1. Ilaç konjugatları hazırlanması <ol><li> 1,5 ml reaksiyon tüp içinde 10 mg 1 ml dH 2 O DF çözülür. Bu DF çözüm daha fazla ilerlemeden 95 ul kullanın. PP konjuge HSA 500 ul 0.3 M sodyum hidroksit (NaOH) 30.4 mg PP türev (304 g / mol) çözülür. </li><li> (N-morfolino) ethanesulfonic asit (MES) tampon pH 6,5 - 95 ul çözünmüş DF - 430,6 ul dH 2 O ve 100 ul 0.5 M 2 ekleyin . 33.1 ul dH 2 O, ve PP örnek 2 M MES tampon pH 2.9 140 ul ekleyin . </li><li> 57.5 taze hazırlanmış N-etil-N &#39;ul ekle DF örnek mg / ml konsantrasyon, ya da 100 10 ul EDC çözüm dH 2 O çözünmüş carbodiimide hidroklorür (EDC) stok solüsyonu (3-Gaz sistemlerinde ) PP örnek. Vortex 1 dakika boyunca örnekleri. </li><li> Her bir örnek için 118 mg / ml &#39;lik bir konsantrasyon ile HSA çözüm 16.9 ul ekleyin. </li><li> 300 mermi / dak sallayarak ederken örnekleri 2 saat oda sıcaklığında inkübe edin. </li><li> DF ve PP örnekleri 3 kez, her biri için birkaç saat fosfat tamponlu salin (PBS) pH 7.4 2 litre karşı Dialyze. 4 ° C&#39;de bütün diyaliz adımları uygulayın. </li><li> Örnek 10 dakika 14.000 xg&#39;de santrifüjleyin ve supernatant içeren DF veya konjuge HSA PP toplamak. </li><li> Proteinleri SDS-PAGE gerçekleştirerek% 12 jeller kullanarak süpernatant kontrol edin. Bir jel yuvaya yaklaşık 12 mikrogram HSA konjuge yükleyin. </li></ol> 2. Ilaç konjugatları kavrama oranı (Şekil 2) belirleyin <ol><li> 150 mM NaCl ve% 0.05 sodyum azid içeren 100 mM sodyum fosfat tamponu pH 6.5 HPSEC DF ve PP konjugatları kaplin oranı analizi için kullanın. TSK-Gel G2000 SWXL sütun bir 7.8 mm x 300 kullanın. </li><li> Online birleştiğinde RI ve UV dedektör sistemi kullanarak sinyallerin tespit gerçekleştirin. </li><li> DH 2 O 1 mg / ml &#39;lik bir konsantrasyon ile HSA standart bir örnek hazırlayın Dedektör kalibrasyonu için 50 ul HSA standart solüsyon enjekte edilir. </li><li> RI sabiti k RI ve dedektör UV sabiti k UV hesaplayın. Formüller alan RI = k RI * [(dn / dc) HSA * c Kalkış Desteği (HSA) ve alan UV = k UV * [(dA / dc) HSA * c Kalkış Desteği (HSA)] (dn / dc) HSA = 0,185 ve (dA / dc) HSA = 0,518 5. </li><li> DF ve her iki standartları, 5, 10, ve 30 nmol miktarda kolaylıkla enjekte edilebilir HPSEC için PP türev standartları hazırlayın. </li><li> (Dn / dc) için ilaç ve her iki standardın (dA / dc) ilaç ortalama değerleri hesaplayın. DF, (dn / dc) 39.000 ± 0.010 (dA / dc) ± 0,493 0,235 tespit edildi. PP, 0.328 ± 0.016 (dn / dc) ve (dA / dc) 53,155 ± 2,464 tespit edildi. </li><li> HPSEC ilaç konjügatları enjekte edilir ve RI ve UV pik alanları belirlemek. </li><li> DF, PP türev, ve HSA konsantrasyonu = k RI * (dn / dc) ilaç * c (ilaç) + (dn / dc) HSA * c Kalkış Desteği (HSA) ve alan UV iki denklemler alanında RI çözme hesaplayın = k UV * [(dA / dc) ilaç * c (ilaç) + (dA / dc) HSA * c] bilinmeyenli Kalkış Desteği (HSA). </li></ol> 3. BAT kullanarak ilaç konjugatları <ol><li> Yedi akış sitometri şişeleri hazırlayın ve içeriklerine bağlı olarak etiket: lekesiz kontrol, negatif kontrol, pozitif kontrol ve ilaç konjugatları. </li><li> B-CCR-STB stimülasyon, lekesiz ve negatif kontrol için ayrılmış şişeleri içine tampon (Flow2 CAST, Buhlmann Laboratuvarları, Schönenbuch, CH) Pipet 50 ul. Pozitif kontrol olarak, bir anti-FcεRI çözüm Flow2 CAST seti tarafından sağlanan 50 ul kullanın. 0.02 -20 mg / ml DF konjuge ve PP konjuge (200 ul bir test hacmi son konsantrasyonları) 1:10 seyreltme serisi oluşturarak, ilaç konjugatları için ayrılmış şişeleri, konjuge çözüm uygun miktarlarda ekleyin. Bu titrasyon deneyleri, her bir hasta numunesi için en uygun bazofil aktivasyon konsantrasyonunu belirlemek için yapılmalıdır. </li><li> Her bir tüp ve 50 ul hasta EDTA tam kan için 100 ul stimülasyon tampon ekleyin. Örnekleri dikkatlice karıştırın. </li><li> Pipet 20 ul Flow2 CAST boyama fikoeritrin (PE) ve her tüp floresein izotiyosiyanat (FITC) ile işaretli anti-CD63 antikorları ile işaretli anti-CCR3 antikorları içeren reaktif ve tekrar karıştırın. Lekesiz numune boyama reaktif pipetle etmeyin! </li><li> 37 ° C&#39;lik su banyosunda 45 dakika örnekleri inkübe edin. </li><li> 5 dakika boyunca buz üzerinde reaksiyon durdurun. </li><li> Her flakon ve vorteks hafifçe 2.0 ml Flow2 CAST parçalama reaktifi eklenerek eritrositler Lyse. Numune karanlık oda sıcaklığında 10 dakika inkübe edin. </li><li> 500 xg&#39;de 5 dakika ve dikkatle boşaltmak süpernatant için numuneleri santrifüjleyin. </li><li> Akış sitometri kadar buz üzerinde 500 ul Flow2 CAST yıkama tamponu ve mağaza hücrelerin tekrar </li><li> Akış sitometresinin gerilim s ayarlayınileri ve yan dağılım (FSC, SSC) yarlar lekesiz örnek elde edilirken. </li><li> Kapısı hücreleri SSC-PE arsa SSC-FSC arsa lenfositler olarak öngördü. Bazofiller CCR3 hi SSC lo ve kapı FITC-PE arsa içine PE etiketli anti-CCR3 antikor tarafından tespit edilir. Aktive bazofiller tespit anti-CD63 antikor FITC etiketli. </li><li> CD63 yüksek negatif kontrol olarak bazofiller cut-off maksimum% 2,5 &#39;i ayarlayın. &gt;% 5 bazofiller CD63 hi ve negatif kontrol MFI bölünmüş örnek ortalama floresan indeksi (MFI) 2 aşarsa (Şekil 3), örnek bir bazofil aktivasyonu için pozitif olarak kabul edilir. </li></ol> 4. Temsilcisi Sonuçlar: Bu deney, IgE bağımlı ilaç aşırı duyarlılık tespit etmek için yararlı bir araç olarak BAT değerlendirir. NSAİİ Protein konjugatları bazofil aktivasyonu için kullanılır. HPSEC olarak RI ve UV saptaması toplama devlet, kaplin derece ve konjugatları etkin verim ile belirlenir. Şekil 4&#39;te görüldüğü gibi, DF konjugatları 43.5% 5.0 DF / HSA kaplin derecesi ile monomerik kaldı. Agrega için 9.5 kaplin derece tespit edildi. Propyphenazone konjuge 68.5% 21.2 kaplin derecesi ile monomerlerin oluştuğu gösterilmiştir. Agregaların kaplin derece 27,9 oldu. DF konjugatları belirlenen kaplin derece toplam 7.6 DF ve PP konjugatları 23.2 idi. BAT IgE bağımlı NSAİİ aşırı duyarlılık reaksiyonları soruşturma izin optimize koşulları (konjugatları konsantrasyonu, stimülasyon süresi) altında konjugatları ile yapıldı. Bazofil% 20,6 aktif floresan bir günlük vardiya ile karakterizedir: Şekil 5 de görüldüğü gibi, sadece PP konjuge bazofil aktivasyonu CD63 yüzey ekspresyonu bir upregülasyonu tarafından görüntülendi tetiklemek mümkün . MFI, 1893 için 4.9 kat artarak 386 (negatif kontrol). Buna karşılık, DF konjuge kullanarak bazofiller sadece% 3.1,% 5 cut-off altında olduğu aktive edildi. Ayrıca, MFI 210 197 (negatif kontrol), 1.1 (sınırı 2,0) bir faktör sadece arttı. Testi geçerliliği tarafından verilen (i) &lt;% 5 negatif kontrol bazofil aktivasyonu, bazofil ICSI&#39;nin floresan yoğunluğu (ii) bir günlük vardiya, ve (iii)&gt;% 50 aktif bazofiller (pozitif kontrol). <img alt="Şekil 1" src="/files/ftp_upload/3263/3263fig1.jpg" /> Şekil 1 HSA DF ve PP türevi çiftlenmesi. Çözüldükten sonra ilaç, su, MES tampon ve EDC (kaplin reaktif) eklenir. HSA ilaç çözümlere pipetlenir önce örnekleri 1 dakika için vortekslenmiş. , Oda sıcaklığında sallayarak 2 saat içinde ilaçlar HSA kovalent bağlanır. Monomerler yanı sıra agrega neden olabilir. <img alt="Şekil 2" src="/files/ftp_upload/3263/3263fig2.jpg" /> Şekil 2 kaplin derece hesaplanması. İlk olarak, RI ve k UV k sabitleri belirlenmiştir. Bu nedenle, bilinen konsantrasyon ile HSA standardı HPSEC sistemi içine enjekte edilir. (Dn / dc) ve HSA (dA / dc) sırasıyla 5, 0,185 ve 0,518 değerleri verilmiştir. Pik alanları sabitleri hesaplamak için kullanılır. İkincisi, her ilaç için üç standartları ilaç ve konsantrasyon-RI ve UV alanları belirlemek için enjekte edilir. Yukarıdaki adımları, k RI ve k UV sabitleri bilinen bu yana, ilaca özgü (dn / dc) ve (dA / dc) türevleri hesaplanabilir . Üçüncü olarak, ilaç-HSA konjugatları HPSEC içine enjekte edilir. Sonuçta elde edilen pik alanları iki bilinmeyen değişkenler ile iki denklemleri çözerek tepe başına ilaç ve HSA konsantrasyonlarını hesaplamak için kullanılır. <img alt="Şekil 3" src="/files/ftp_upload/3263/3263fig3.jpg" /> Şekil 3 bazofil yolluk. Lenfositleri forward scatter karşı kapılı yan dağılım (SSC-FSC arsa) gibi hücre öngördü. Bazofiller CCR3 hi SSC lo kapılı lenfositler (SSC CCR3-PE arsa) olarak tanımlanır. Bazofil aktivasyon (CD63 hi), CD63-FITC-CCR3-PE arsa analiz edilmektedir. Negatif kontrol CD63 hi kalan maksimum% 2.5 bazofiller cut-off ayarlamak için kullanılır. <img alt="Şekil 4" src="/files/ftp_upload/3263/3263fig4.jpg" /> Şekil 4 ilaç konjugatları kaplin derece kararlı. RI ve UV sinyallerinin ilaç HSA konjugatları agrega (düşük tutma hacmi salınımlı) ve monomerlerin (yüksek tutma hacmi az salınımlı) temsil eden iki tepe göstermektedir. Monomerlerin ve agrega yüzdesi (RI sinyalleri tespit) ve kaplin derece (n = 1) tasvir edilmektedir. <img alt="Şekil 5" src="/files/ftp_upload/3263/3263fig5.jpg" /> Şekil 5 bazofil activatitest. CD63-FITC-CCR3-PE araziler (n = 1) ilaç bileşimi ile aktive örnekleri, negatif kontrol, pozitif kontrol sonuçları gösterilmiştir.

<ol>
	<li>Himly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgE-mediated+immediate-type+hypersensitivity+to+the+pyrazolone+drug+propyphenazone.">IgE-mediated immediate-type hypersensitivity to the pyrazolone drug propyphenazone.</a> J Allergy Clin Immunol. 111, 882-888 (2003).</li><li>Demoly, P., Pichler, W., Pirmohamed, M., Romano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+questions+in+Allergy:+1--drug+allergy/hypersensitivity.">Important questions in Allergy: 1--drug allergy/hypersensitivity.</a> Allergy. 63, 616-619 (2008).</li><li>Ebo, D. G., Hagendorens, M. M., Bridts, C. H., Schuerwegh, A. J., Clerck, L. S., Stevens, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+in+vitro+activated+basophils,+specific+IgE+and+skin+tests+in+the+diagnosis+of+pollen-associated+food+allergy.">Flow cytometric analysis of in vitro activated basophils, specific IgE and skin tests in the diagnosis of pollen-associated food allergy.</a> Cytometry Part B (Clinical Cytometry). 64B, (2005).</li><li>Harrer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diclofenac+hypersensitivity:+antibody+responses+to+the+parent+drug+and+relevant+metabolites.">Diclofenac hypersensitivity: antibody responses to the parent drug and relevant metabolites.</a> PLoS One. 5, e13707-e13707 (2010).</li><li>Wen, J., Arakawa, T., Philo, 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=Size-exclusion+chromatography+with+on-line+light-scattering,+absorbance,+and+refractive+index+detectors+for+studying+proteins+and+their+interactions.">Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions.</a> Anal Biochem. 240, 155-166 (1996).</li><li>De Weck, A. L., Sanz, M. L., Gamboa, P. M., Aberer, W., Bienvenu, J., Blanca, M., Demoly, P., Ebo, D. G., Mayorga, L., Monneret, G., Sainte Laudy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+tests+based+on+human+basophils:+more+potentials+and+perspectives+than+pitfalls.+II.+Technical+issues.">Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls. II. Technical issues.</a> J Investig Allergol Clin Immunol. 18, 143-155 (2008).</li><li>Ebo, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-assisted+allergy+diagnosis:+current+applications+and+future+perspectives.">Flow-assisted allergy diagnosis: current applications and future perspectives.</a> Allergy. 61, 1028-1039 (2006).</li><li>Poulsen, L. K., Quan, S., Kragh, C., Platzer, M. H., Skov, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basophil+granulocyte+in+allergic+reactions:+experimental+models+and+their+use+for+the+identification+of+drugs+with+effects+or+side+effects+on+basophils.">The basophil granulocyte in allergic reactions: experimental models and their use for the identification of drugs with effects or side effects on basophils.</a> Curr Med Chem - Anti-inflammatory &#38; Anti-Allergy Agents. 3, 167-180 (2004).</li><li>Sanz, M. L., Gamboa, P., de Weck, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+combined+test+with+flowcytometric+basophil+activation+and+determination+of+sulfidoleukotrienes+is+useful+for+in+vitro+diagnosis+of+hypersensitivity+to+aspirin+and+other+nonsteroidal+anti-inflammatory+drugs.">A new combined test with flowcytometric basophil activation and determination of sulfidoleukotrienes is useful for in vitro diagnosis of hypersensitivity to aspirin and other nonsteroidal anti-inflammatory drugs.</a> Int Arch Allergy Immunol. 136, 58-72 (2005).</li></ol>

Çıkar çatışması ilan etti.

BAT iyi kurulmuş henüz IgE aracılı allerjik hastalık 6,7 tanısı için rutin olarak kullanılan yöntem olsa da. Ilaç aşırı duyarlılık için düşük molekül ağırlıklı bileşikler çapraz IgE reseptörleri, bazofil aktivasyon 8 için bir ön koşul değildir, ancak, bunun uygulanabilirliği tehlikeye düşer. Bu nedenle, soruşturma altında ilaçların kovalent uygun taşıyıcı proteinler (örneğin HSA) ile birleştiğinde olması gerekir. Daha da önemlisi, kaplin derecesi (yani taşıyıcı protein başına ilaç moleküllerinin sayısı) konjugatları (yani IgE reseptörü cross-linking) immünolojik aktivite garanti etmek için kontrol edilmesi gerekiyor. Teorik olarak, taşıyıcı molekül başına iki hapten IgE reseptörü için yeterli olmalıdır çapraz bağlama ve HSA başına beş DF molekülleri gibi birkaç hücre bazlı analiz 4 mediatör salınımını tetikler görülmüştür. Her iki konjugatları, PP-HSA ve DF-HSA, yeterince yüksek bir bağlantı derecesi (HPSEC) gösterilecek ve onlara BAT kullanım için uygun reaktifler immünolojik etkin olacak şekilde tespit edilmiştir. Bir diğer sorun, ana ilacın yerine metaboliti olarak aşırı duyarlılık reaksiyonuna neden olabilir, ilaç metabolitleri bir rol oynayabilir olabilir. DF durumunda, bu olasılığı daha önce beş büyük Faz I metabolitleri ve bir bağ varyant 4 kullanılarak ayrıntılı olarak değerlendirilmiştir . Açıklanan teknik mastering için önemli faktörler kan örneği (&lt;kan toplama, tespit bazofiller sayısı bu yana 12 saat&gt; 500) ve stimülasyon koşullarının optimizasyonu (zaman, doz) kalite içerir. Buna ek olarak, bu bile pozitif kontrol (antikor IgE reseptörü yöneltilen) tespit edilmesi ile reaksiyona girmeyen olmayan-yanıt olarak adlandırılır. Bu nedenle, doğrulama kriterleri, pozitif kontrol olarak bir anti-FcεRI antikor dahil etmek zorunda. Ilaç konjugatları kullanarak önemli bir avantajı DF-HSA konjuge anti-BAT 4 FcεRI antikor ile birlikte stimülasyon için gösterilen saf ilaçlar, aksine non-toksik ortaya çıktığı bir gerçektir. Saf DF, aksine, 1.25 mg / ml konsantrasyonda BAT 9 yorumlanabilirliğini sorunlara neden sitotoksik etkileri gösterir. Genel olarak, potansiyel sitotoksisite test kuvvetle her yeni üretilen ilaç bileşimi için tavsiye edilir. Burada, BAT IgE aracılı aşırı duyarlılık ortaya çıkarmak için kullanılan PP-HSA konjuge potansiyel göstermiştir. Buna karşılık, DF aşırı duyarlılık DF-HSA konjuge yanıt bazofil aktivasyon eksikliği gösterdiği IgE ile ilişkili değildir. Önemlisi, IgE aracılı bir mekanizma ile ilgili belirsizlik durumunda, konjugatları DF 4 gösterildiği gibi, in vitro hücre tabanlı test sistemleri immünolojik aktivite için değerlendirilmek üzere. Uygun protein taşıyıcı kovalent düşük molekül ağırlıklı ilaçların conjugating açıklanan kurulumu kullanarak, antibiyotikler de dahil olmak üzere ilaç, diğer NSAID, radiocontrast medya, kas gevşeticiler, anestezi karşı aşırı duyarlılık reaksiyonları bir dizi moleküller, vb IgE aracılıklı bir katılımı için soruşturma olabilir mekanizması. Bu nedenle, BAT tanı (cilt testi, oral provokasyon testi) mevcut yöntemlere ek olarak hizmet verebilir.

<table border="1"><tbody><tr><th> Reaktif / cihaz Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> 2 - (N-morfolino) ethanesulfonic asit </td><td> Sigma </td><td> M3671 </td><td> - </td></tr><tr><td> 7.8 x 300 mm TSK Jel -G2000SWXL sütun </td><td> Bioscience Tosoh </td><td> 08540 </td><td> - </td></tr><tr><td> BD FACSCanto II. </td><td> Becton Dickinson </td><td> 338962 </td><td> - </td></tr><tr><td> Masa üstü santrifüj </td><td> Sigma </td><td> - </td><td> - </td></tr><tr><td> Diklofenak sodyum tuzu </td><td> Sigma </td><td> D6899 </td><td> - </td></tr><tr><td> EDTA-Vacutainer </td><td> Becton Dickinson </td><td> 368589 </td><td> - </td></tr><tr><td> Flow2 CAST Seti </td><td> Buhlmann Laboratuvarları </td><td> FK-CCR </td><td> Etkili 14 Haziran 2011 Flow2 CAST kiti Akış CAST </td></tr><tr><td> HP1100 HPLC sistemi </td><td> Hewlett Packard </td><td> - </td><td> - </td></tr><tr><td> İnsan Serum Albumin </td><td> Sigma </td><td> A9511 </td><td> - </td></tr><tr><td> Laboratuvar Santrifüj </td><td> Eppendorf </td><td> - </td><td> - </td></tr><tr><td> N-etil-N&#39;-(3-Gaz sistemlerinde) carbodiimide hidroklorür (EDC) </td><td> Sigma </td><td> E6383 </td><td> - </td></tr><tr><td> PBS tablet </td><td> ¸ </td><td> A9199 </td><td> - </td></tr><tr><td> Propyphenazone türevi </td><td> Moleküler Biyoloji Anabilim Dalı, Salzburg Üniversitesi, Avusturya </td><td> - </td><td> - </td></tr><tr><td> Shaker </td><td> Eppendorf </td><td> - </td><td> - </td></tr><tr><td> Sodyum azid </td><td> Sigma </td><td> S8032 </td><td> - </td></tr><tr><td> Sodyum klorür (NaCl) </td><td> Sigma </td><td> S1679 </td><td> - </td></tr><tr><td> Sodyum hidrojen karbonat </td><td> Sigma </td><td> 90421C </td><td> - </td></tr><tr><td> Sodyum hydroxid </td><td> Sigma </td><td> S5881 </td><td> - </td></tr><tr><td> Sodyum fosfat </td><td> Sigma </td><td> 342483 </td><td> - </td></tr><tr><td> Üçlü dedektör dizi </td><td> Viscotek </td><td> TDA302 </td><td> - </td></tr><tr><td> Kişisel BioVortex V-1 artı </td><td> Peqlab </td><td> 90-V-1 </td><td> - </td></tr><tr><td> Su banyosu 1012 </td><td> GFL </td><td> 1012 </td><td> - </td></tr></tbody></table>

basophil activation test for investigation of ige-mediated mechanisms in drug hypersensitivity

Propyphenazone (PP) ve diklofenak (DF) gibi non-steroid anti-enflamatuar ilaçlar (NSAID) karşı aşırı duyarlılık reaksiyonları, alerjik reaksiyonlar 1 I-benzeri türü olarak tezahür edebilir . Klinik uygulamada, ilaç aşırı duyarlılık tanısı deri testleri güvenilir değildir ve oral provokasyon testi 2 hasta için hayatı tehdit eden riskler taşımaktadır esas olarak hastanın öyküsü tarafından yapılır. Dolayısıyla, altta yatan bir IgE aracılı pathomechanism delil elde etmek zordur. Burada, in vitro yöntem, ilaca duyarlı hastalarda in vivo olarak, alerjik efektör reaksiyon taklit edilen insan bazofiller kullanımına dayalı bir sunuyoruz. Ilaç alerjik hastalarda bazofil suçlu ilaç için spesifik IgE molekülleri taşıyan, IgE reseptörü crosslinking üzerine aktif ve alerjik effektör moleküllerin serbest hale gelir. Bazofil aktivasyonu CD63 yüzey ekspresyonu flow sitometri 3 kullanarak upregülasyonu belirlenmesi ile takip edilebilir. Düşük molekül ağırlıklı ilaçların durumunda, konjugatları bazofiller IgE reseptörü crosslinking sağlamak için tasarlanmıştır. Şekil 1, iki temsilci NSAİİ tasvir gibi, PP ve DF, bir karboksil grubu ile lizin kalıntılarının temel amino grubu ile reaksiyona Kalkış Desteği (HSA) insan serum albumini için kovalent bağlıdırlar. DF, bir karboksil grubu içeren PP türevi organochemically önce çalışma 1 sentezlenebilir sahipken, bu nedenle, doğrudan 4 kullanılabilir içsel bir karboksil grubu ve taşımaktadır . Iki IgE reseptör molekülleri crosslinking garanti etmek için protein taşıyıcı molekül düşük molekül ağırlıklı bileşikler ve mekansal dağılımının kaplin derece önemlidir. Burada açıklanan protokol sıralı bir kırılma indeksi (RI) ve ultraviyole (UV) kaplin derecesinin belirlenmesi için algılama sistemi ile donatılmış yüksek performans boyut dışlama kromatografisi (HPSEC) geçerlidir. Açıklanan metodoloji diğer ilaçlar için uygulanan olabilir, bazofil aktivasyon testi (BAT), ilaç aşırı duyarlılık IgE aracılı mekanizmaların belirlenmesi için kullanılan olma potansiyeli taşımaktadır. Burada, PP aşırı duyarlılık IgE aracılı ve DF aşırı duyarlılık, BAT, non-IgE aracılı olarak belirlemek.

Watch this Scientific Journal Video about Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity at JoVE.com

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Bazofil aktivasyon testi, IgE bağımlı alerji tespiti için güçlü bir araçtır<em&gt; In vitro</em&gt;. Burada, bazofil aktivasyon testi için optimize edilmiş bir protokol ilaç aşırı duyarlılık araştırmak için kullanılır. Kovalent ilaç-protein konjugatları ve fizikokimyasal karakterizasyonu ve verimli üretim için bir yöntem açıklanmıştır.

İlaç Aşırı duyarlılık IgE aracılığı ile Mekanizmalarının Araştırılması bazofil Etkinleştirme Test

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

basophil-activation-test-for-investigation-ige-mediated-mechanisms

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33.3K Görüntüleme.  University of Salzburg. Bazofil aktivasyon testi, IgE bağımlı alerji tespiti için güçlü bir araçtır In vitro. Burada, bazofil aktivasyon testi için optimize edilmiş bir protokol ilaç aşırı duyarlılık araştırmak için kullanılır. Kovalent ilaç-protein konjugatları ve fizikokimyasal karakterizasyonu ve verimli üretim için bir yöntem açıklanmıştır.

Video: Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Murin Model B hücreleri CD40 aktivasyonu

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

İmmünoloji ve Enfeksiyon

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Kolaylaştırılması İlaç Keşfi: Zebra balığı An Otomatik Yüksek içeriği Enflamasyon Assay

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Antijen Özgül Düzenleme için Trans-vivo Gecikmeli Tip Aşırı Duyarlılık Testi

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Aktivasyon ve İnsan monosit türevli dendritik hücreler, IL-1β kullanarak NLRP3 Inflammasome Aktivitenin Ölçümü

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T Lenfositlerindeki Integrin Aktivasyonunun Çalışması için statik Adezyon Deneyi

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Yüksek verim Kantitatif Gerçek zamanlı RT-PCR Testi Türleri I ve III İnterferon Alttipleriyle İfadesi Profilleri belirlenmesi için

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

Çok ilaca dirençli için Antimikrobiyal Mavi Işık Tedavisinin Vivo İncelenmesinin Acinetobacter baumannii Biyoparlaklık ımaging&#39;i kullanarak Enfeksiyonlar Yanık

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Patojenik Mekanizmalarının İncelenmesi için BALB/c Farelerde İlaca Bağlı, Otoimmün Hepatitin İndüklenmesi

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

In Vitro ELISA Testi Kuduz Aşısı Potens Değerlendirmek için

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.