Name: detection of true ige-expressing mouse b lineage cells
Uploaded: 2014-12-01T14:00:00.000+00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

DRW NIH hibe AI089972 ve AI113217 tarafından ve Mukozal İmmünoloji Çalışmaları Ekibi tarafından desteklenen ve Burroughs Wellcome Fonu Tıbbi Bilimciler için Kariyer Ödülü tutar.

NOT: Burada açıklanan tüm deneyler Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) kurallarına uygun olarak ve Hayvancılık Araştırma Hastanesi Çocuk (ARCH), Boston, Mass tarafından onayladı. Reaktifler 1. Hazırlık <ol><li> 1,000x, IL-4 Stok: H 2, O ya da% 0.1 bovin serum albümini (BSA) içinde 20 ug / ml olacak şekilde seyreltin, IL-4, sitokin. -20 ° C&#39;de 1.5 ml mikrosantrifüj tüplerine ve saklamak için 200 ul alikolar içinde dağıtın. </li><li> 1,000x anti-CD40: 4 ° C &#39;de, 1.0 mg / ml arasında, mağazada üreticisinden elde edilir. </li><li> B hücre uyarılmasını ortamı: RPMI-1640 ortamı içinde 425 ml&#39;lik, ekleme 75 taze çözülmüş fetal buzağı serumu mi (FCS), 1.0 M HEPES tamponunda, 5 ml L-glutamin stokunun 10 mi, 5 ml Penisilin: Streptomisin stoku, 5 2-merkaptoetanol mi Asgari Temel Ortam Aslî Olmayan Amino Asitler (MEM-NEAA) ve 3.5 ul. Bir haftaya kadar 4 ˚C Ortamı tutun. IL-4 (20 ng / ml) ve anti-CD40 (1.0 ug / ml), kullanılmadan hemen önce eklemesadece gerekli ortamın hacmi. </li><li> % 2 FCS-FACS Tampon: 1x PBS 490 ml FCS, 10 ml ilave edilir. 4 ° C&#39;de tutun. </li><li> 2x tripsin-EDTA çözeltisi: 1x PBS, 20 ml 10 x tripsin-EDTA stok çözeltisi (5.0 gr, tripsin, litre başına 2.0 g EDTA) 5 ml seyreltin. Mağaza tripsin-EDTA 2x 4 ° C&#39;de alikotları çalışma. Kullanmadan önce oda sıcaklığına ısınmasına izin verin. </li><li> Cenin buzağı serumu: en fazla iki hafta boyunca 4 ° C&#39;de eritilmiş FCS tutun. Sitoplazmik boyama prosedürü önce buz 15 dk yerleştirin. </li><li> Nötr-tamponlu formalin:% 10 formalin solüsyonu (% 3.7 paraformaldehid) hazırlayın. Formalin toksik ve uygun KKD giyerek bu nedenle laboratuar davlumbaz altında tutun. Bir kimyasal depolama kabini oda sıcaklığında saklayın formalin. </li><li> Metanol: -20 ° C&#39;de saklayın mutlak metanol (% 100) hücreleri geçirgenleştirildi hazır olana kadar. Bu, metanol yanıcı ve uygun bir dondurucuda saklanabilir gerektiğine dikkat ediniz. </li></ol> 2.Saflaştırılması ve B hücrelerinin aktivasyonu <ol><li> Taze hasat fare dalak kaynaktan, dikkatli bir şekilde 3 ml lik bir enjektör pistonuna sahip 70 mikron hücre süzgecinden organı basarak tek bir hücre süspansiyonu hazırlanır. 15 ml konik santrifüj tüpü içinde, 4 ° C&#39;de 5 dakika boyunca 300 x g&#39;de 1x 10 ml PBS hücrelerin ve santrifüj toplar. </li><li> 5 dakika boyunca alyuvar parçalanması Tamponu, 1 ml yüzer madde ve tekrar süspansiyon pelet süzün. 5 dakika boyunca 300 xg&#39;de 13.5 1x PBS ml ve santrifüj ile nötralize edin. </li><li> (İsteğe bağlı) Manyetik üreticinin talimatlarına göre MACS arıtma protokolü takip edilerek, boncuklar, anti-CD45R (B220) ile B hücreleri -etiketli ayırın. </li><li> 37 ° C &#39;de, IL-4 (20 ng / ml) ve 5 gün boyunca bir anti-CD40 (1.0 ug / ml) ihtiva eden ısıtılmış B hücre uyarılmasını ortam içinde 1.0 x 10 6 hücre / ml&#39;de, saflaştırılmış B hücrelerini stimüle eder. 6 oyuklu bir kültür plakasına (4 mL) içinde 2 kuyu ayrılmıştır kültür 8 ml ile başlar. </li><li> Cultu edin1.0 x 10 6 hücre / ml&#39;de korumak için günlük olarak tekrar. Diğer kuyulara bölme ve taze IL-4 ve anti-CD40 ihtiva eden B hücre uyarılmasını ortamı ile kültür seyreltilerek konsantrasyonu ayarlayın. </li></ol> 3. tripsinizasyonu, Fiksasyon ve Permeabilization <ol><li> Trypsinization <ol><li> 5 dakika boyunca 300 xg&#39;de, 15 ml konik tüp içinde santrifüj kültür hücrelerinin kadar 1.0 x 10 7 toplayın, ve süpernatant süzün. </li><li> Hücre toplama ortamından artık proteinin ayrılması için 10 ml PBS ile yıkayın. Santrifüj adımı tekrarlayın ve süpernatant süzün. </li><li> (Yaklaşık 100 ul) boşaltılarak sonra rezidüel hacim hücre pelletini. </li><li> Dikkatlice hücreler, oda sıcaklığında,% 0.1 (2x) tripsin-EDTA, 800 ul ilave edin. Konik tüp tarafında aşağı 45 ° açıyla yavaşça koyun. </li><li> Tüp kapatın ve sıvı tüp 5-6 kez uzunluğu boyunca uzanan bu yüzden tüp eğerek hafifçe karıştırıns. Bir tel, beyaz bir çökelti çözeltisi içinde meydana gelebilir. Bir dakika altındaki trypsinization zamanı sınırlayın. </li><li> Soğuk FCS, 1 ml tripsinizasyon reaksiyonu söndürün. Hemen soğuk PBS 10 ml ilave etmek suretiyle FCS ile seyreltilir. </li><li> 4 ° C&#39;de 5 dakika boyunca 300 x g&#39;de santrifüj, ardından süpernatan süzün ve buz tüpleri döndürür. </li></ol></li><li> Tespit <ol><li> Boşaltılarak sonra rezidüel hacim hücrelerin tekrar. Gerekirse, soğuk PBS ile 100 ul hacmi getirmek. </li><li> Bir çeker ocak içinde, hücrelere% 10 nötr tamponlu formalin çözeltisi 800 ul ve yukarı ve aşağı pipetleme ve. Tüp formalin ilavesi üzerine sıcak olacak. 10 dakika boyunca 37 ° C&#39;de inkübe edilir. </li></ol></li><li> Permeabilizasyon <ol><li> Hafif sallayarak hızında bir tezgah üstü vorteks karıştırıcı ayarlayın. </li><li> 10 ml&#39;lik bir serolojik pipet içine soğuk (-20 ° C) metanol içinde 9 ml çizin. </li><li> Vorteks mikser üzerinde hücreleri içeren tüp tutun ve yavaşça sallıyor başlangıçg kadar hücre. </li><li> Tüp hala çok titriyor metanol sürekli karıştırma olduğunu iken hücreleri üzerine damla damla düşer, böylece tüp soğuk metanol ekleyin. Metanol ilk 2 ml sonra dağıtma hızı yavaş bir akışla kadar arttırılabilir. </li><li> 30 dk permeabilization tamamlamak için tüp buz üzerinde bekletin. Seçenek olarak ise, hücreler, bir aya kadar -20 ° C&#39;de muhafaza edilebilir. </li></ol></li></ol> IgG1 ve IgE sınıf değiştirme 4. Floresan Boyama <ol><li> Santrifüj 5 dakika boyunca 300 xg&#39;de örnek ve metanol çözüm süzün. Beyaz bir çökelti hücreleri ile pelet olabilir. </li><li> 5 dakika Hücreleri yıkamak için 300 x g&#39;de oda sıcaklığında PBS 1x ve santrifüj 10 ml ilave edilir. Kez tekrarlayın. Not: oda sıcaklığı PBS ile pelet yıkanması adım 4.1 sonra oluşabilecek ekstra çökelti çözer. </li><li> % 2 FCS-FACS tamponu 10 ml ek yıkama yapın. </li><li> Yuvarlak tabanlı plaka 96 çukurlu numuneleri dağıtmave 5 dakika boyunca 300 x g&#39;de santrifüj. Süpernatant süzün ve buz üzerinde tabak koydu. </li><li> Aşağıdaki antikor seyreltmeleri ile oyuk başına% 2 FCS-FACS tamponu 60 ul Leke: Anti-IgM RPE / Cy7, 1: 200, anti-lgG1 PE, 1: 600, anti-IgE FITC, 1: 100, Anti- CD45R (B220) PerCP Cy5.5 @, 1: 200 </li><li> Boyama 5 dakika sonra, 300 x g, 5 dakika boyunca% 2 FCS-FACS tamponu ve santrifüj 150 ul plakayı yıkayın. </li><li> 5 ml&#39;lik bir FACS tüpü içine, bir 40 um naylon hücre süzgecinden% 2 FCS-FACS tamponu ve pipet 400 ul içinde süspanse. </li></ol> 5. Aşağıdaki Yolluk Strateji kullanın: <ol><li> FSC / SSC arsa (Şekil 1) bulunan patlatma lenfosit nüfus CD45R (B220) pozitif hücreleri izole. </li><li> B220 pozitif hücrelerden, ilgili popülasyonları ortaya IgE ve IgG1 arsa kullanın. </li></ol>

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Anti-CD40 ve IL-4 ile kültürde fare dalak B hücreleri uyarımı IgG1 ve IgE 5, T yardımcı tip 2 (Th2) etkileşimi teşvik edici sınıf değiştirme simule eder. B hücreleri, toplam splenositler 12 ya da saflaştırılmış ve splenik B hücrelerinin 11 bağlamında CSR etkinleştirilebilir. Protokol (adım 2.3) de belirtildiği gibi, B hücresi zenginleştirme isteğe bağlıdır, ve yararlı olurdu olmadığını belirlemek için deneyci takdirine bağlıdır. CD45R (B...

Fare ve insan immünoglobulin ağır zinciri (IgH) sınıf anahtarı rekombinasyon (CSR) sırasında, IgH, sabit bölge ekzon (Cμ) silindi ve aşağı doğru sabit bölge eksonların çeşitli kümelerinin bir (Cı lH ler) (örneğin, Cγ değiştirilir μ Ig sınıflarına (örneğin, IgG, IgE ya da IgA) üretimine IgM üretimi ile ilgili bir anahtar elde Cε ve Ca&#39;sına). CSR CH s, 1 her bir grubu için 5 &#39;bölgesinde yer 1-10 kb sekansları anahtarı (s) bölgeleri içinde gerçekleşir. Aktivasyon Bağlı Sıtıdın Deaminaz (AID) enzim sitidinin deaminasyon etkinliği yoluyla CSR başlatır. IgE allerjik hastalık 2 anahtar arabulucu ve IgE üretilen ve atopik hastalığı için yeni tedavi yaklaşımları kapılarını açabilir düzenlenir nasıl büyük bir anlayıştır. Farelerde ve insanlarda olarak IgE, en sıkı bir şekilde düzenlenmiş Ig izotiptir. IgE normal tim düzeyleri binlerce tespit edilires diğer IgH daha az 3 isotypes, ama çok hastalık durumlarında 4 yükselmiş olabilir. Bununla birlikte, IgE CSR tam olarak anlaşılamamıştır. B hücrelerinin in vitro aktivasyonu, IL-4, anti-CD40 veya LPS ile kombinasyon halinde her iki IgG1 ve IgE 5 CSR indükler kullanılmıştır. Aktive B hücreleri çözünebilir IgE CSR sonra kültür salgılanan bağlayan düşük afiniteli IgE reseptörü FcεRII / CD23 2,6, ifade eder. Akış sitometrisi ile analiz, bu nedenle, reseptör bağlı IgE lekesi ile B hücreleri benzer endojen IgE 7 sentezleyen B hücreleri için. O fare B biliniyor olsa da hücreler IgG1 sınıf anahtarlama ölçümü ile müdahale görünmüyor deneyimlerimiz düşük afinite IgG reseptörü FcγRIIB1 (CD32) 8, ifade eder. Kültür içinde, B hücresi aktivasyonu sonrası IgE geçiş ölçüm Ancak, spesifik olmayan yüzeyine bağlı IgE analizi gizleyebilir. Yaygın boyama yöntemleriyle olmayan IgE ifade eden hücreler IgE için pozitif leke. Burada özetlenen fareler 9 arası gerçek IgE ifade B hücreleri KSS tahlilleri yapmak ve tespit etmek için kullanılmıştır bir stratejidir - 11. Tripsin ile aktive B hücrelerinin tedavisi, ortak bir laboratuar reaktif protein sindirimi, cytophylic ve membran hem de bağlı yüzey Ig kaldırır. Sitoplazmik IgE için sonraki permeabilization ve boyama, böylece gerçek IgE üreten hücreleri ortaya koymaktadır.

<table><tbody><tr><td>Anti-Mouse IgE FITC</td><td>BD Pharmingen</td><td>553415</td><td>clone R35-72</td></tr><tr><td>Rat Anti-Mouse IgG1 PE</td><td>BD Pharmingen</td><td>550083</td><td>clone A85-1</td></tr><tr><td>Methanol</td><td>BDH</td><td>BDH1135-4LP</td><td>Keep at -20 &amp;deg;C</td></tr><tr><td>Falcon Cell Strainer 40 &amp;micro;m Nylon</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>Falcon Cell Strainer 70 &amp;micro;m Nylon</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5</td><td>eBioscience</td><td>45-4052-82</td><td>clone RA3-6B2</td></tr><tr><td>anti-Mouse/Rat CD40</td><td>eBioscience</td><td>16-0402-85</td><td>clone HM40-3</td></tr><tr><td>RPMI Medium 1640</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>HEPES (1M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>MEM-NEAA (100x)</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Phosphate Buffered Saline (10x)</td><td>Lifetechnologies (Corning)</td><td>46-013-CM</td><td></td></tr><tr><td>MACS CD45R (B220) microbeads&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-049-501</td><td></td></tr><tr><td>MACS purification column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>IL-4</td><td>PeproTech</td><td>214-14</td><td>Reconstitute in water or 0.1% BSA</td></tr><tr><td>Formalin Solution, Neutral Buffered, 10%</td><td>Sigma Aldrich</td><td>HT501128-4L</td><td></td></tr><tr><td>Trypsin-EDTA Solution (10x)</td><td>Sigma Aldrich</td><td>T4174-100 ml</td><td>5.0 g Trypsin, 2.0 g EDTA&amp;#8226;4 Na per Liter of 0.9% NaCl</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma Aldrich</td><td>P0781-100 ml</td><td>10,000 U Penicillin; 10 mg/ml Streptomycin (100x)</td></tr><tr><td>L-Glutamine 200 mM</td><td>Sigma Aldrich</td><td>G7513</td><td></td></tr><tr><td>2-mercaptoethanol (99%)</td><td>Sigma Aldrich</td><td>M6250-100 ml</td><td></td></tr><tr><td>Red cell lysis buffer</td><td>Sigma Aldrich</td><td>R7757</td><td></td></tr><tr><td>Rat Anti-Mouse IgM RPE/Cy7</td><td>SouthernBiotech</td><td>1140-17</td><td>clone 1B4B1</td></tr><tr><td>HyClone Fetal Calf Serum</td><td>Thermo</td><td>SH30910.03</td><td></td></tr><tr><td>B cell Stimulation media</td><td></td><td></td><td>B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1x MEM-NEAA, 2.0 mM L-glutamine,&amp;nbsp; 1x Penicillin-Streptomycin (Penicillin: 100 U/ml, Streptomycin: 100 &amp;micro;g/ml), Beta-mercaptoethanol (7 &amp;micro;L/L), IL-4 (20 ng/ml), and anti-CD40 (1 &amp;micro;g/ml)</td></tr></tbody></table>

detection of true ige-expressing mouse b lineage cells

B lenfosit immünoglobulin ağır zinciri (IgH) sınıf anahtarı rekombinasyon (CSR) ilk olarak, IgM IgA, IgE ve IgG olarak diğer IgH izotiplerinden geçer ifade edildiği bir süreçtir. In vitro IgH CSR ölçümü, moleküler ve hücresel immünoloji yönlerine DNA yeniden birleştirmesinden ve onarım kadar biyolojik süreçlerin bir dizi çalışma için bir temel yöntem. In vitro CSR deney aktive B hücreleri üzerindeki akış sitometrik ölçüm yüzey Ig ifade içerir. IgA ve IgG alt sınıflarının ölçümü kolay olmakla birlikte, bu yöntemle IgE ölçümü, çözünür IgE FcεRII bağlanma nedeniyle sorunludur / CD23 aktive B hücrelerinin yüzeyi üzerinde ifade edilmiştir. Burada kültür CSR geçirmiş fare B hücreleri, IgE üreten doğru bir şekilde ölçülmesi için benzersiz bir işlem tarif eder. yöntem cy ile sağlanan B tipi hücrelerin IgE üreten tespiti için izin hücre yüzeyleri üzerindeki IgE-CD23 komplekslerinin tripsin-sebepli bölünme dayanırtoplasmic lekelenmesi. Bu prosedür IgE KSS akış sitometri analizi için uygun bir çözüm sunuyor.

Bu prosedür başarıyla fare B hücrelerinde IgE CSR çalışma uygulamaya konmuştur. Daha önce tarif edildiği gibi 11 CSR ölçüm etkinliği tespit etmek için, anti-CD40 ve IL-4 ile birlikte fare dalak B hücreleri stimüle. Stimülasyon beş gün sonra, hücreler toplandı ve protokol floresan-etiketli IgM, IgG1, IgE ve B220 (CD45R) antikorlar ile, yukarıda tarif edilen ve lekeli kullanılarak işlenmiştir. (Şekil 1), IgE + hücrelerin net bir nüfusu temiz tripsin tedavi (...

Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

Detection of True IgE-expressing Mouse B Lineage Cells

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Doğru Algılama Fare B Lineage Hücreleri IgE-ifade

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

detection-of-true-ige-expressing-mouse-b-lineage-cells

Research

JoVE Journal

Immunology and Infection

10.9K Görüntüleme.  Brigham and Women's Hospital and Harvard Medical School. In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Video: Detection of True IgE-expressing Mouse B Lineage Cells

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Sınıf Anahtarı Rekombinasyon murin B Hücreleri Saf İndüksiyon ve Değerlendirme

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

İmmünoloji ve Enfeksiyon

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37&deg;C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent   a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37&deg;C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.

This procedure describes the detection and isolation of mouse TH17 leukocytes that actively secrete IL-17 upon stimulation.

Canlı Fare IL-17 salgılayan T Hücreler Algılama ve İzolasyonu

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

Biyoloji

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Fenotip ve Fonksiyon Analizi için algılama ve Nadir zenginleştirilmesi antijene özgü B hücreleri

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

C57BL / 6 FARELERDE SLE bm12 İndüklenebilir Modeli (SLE)

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

Tıp

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

CD71/TER119 Flow-sitometrik Testi kullanarak Fare eritro-id progenitörlerin Tanımlama ve Analiz

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Timus bağlı ve timus bağımsız immünglobulin izotip yanıt Immunosorbent Assay enzim bağlı kullanarak farelerde karakterizasyonu

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

Murine B-1a Hücrelerinde CXCR4'ün Retroviral Aşırı Ekspresyonu ve Hedeflenen B-1a Hücre Nakliiçin Kemik İliği ve IgM Üretimine Geçişinde Benimsenmiş Transfer

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Murine B Hücre Gelişiminin Akış Sitometrik Karakterizasyonu

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

Murin Akciğerinin Doğuştan Gelen ve Adaptif İmmün Hücrelerinin Tanımlanması için Akım Sitometrik Analizi

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

CD226 Ekspresyonunu Saptamak için Grup 2 Doğuştan Gelen Lenfoid Hücrelerin Fare Burun Mukozasından İzolasyonu

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various ...