Yazarlar kendi teknik yardım için Bing Zhang teşekkür etmek istiyorum. Bu çalışma Sağlık Araştırması (MOP-86595) için Kanada Enstitüleri tarafından finanse edildi. SEKME CIHR Yeni Araştırmacı ve AHFMR Scholar olduğunu. Doktora ve AIHS Tam zamanlı Bursu - HY bir CIHR Kanada Lisansüstü Burs tarafından desteklenmektedir. SAN Kraliçe Elizabeth II Lisansüstü Burs tarafından desteklenmektedir. Doktora - AYWS bir NSERC Lisansüstü Burs tarafından desteklenmektedir.

Deneysel protokol genel bir şema için Şekil 1 &#39;e bakınız. 1. Teşrih <ol><li> 60 x 15 mm Petri kabı içine yerleştirin steril çelik örgü ekran. Bir birim doku örneği başına gereklidir. </li><li> Her bir tabak için Hank&#39;in dengeli tuz çözeltisi (HBSS) 5 ml ilave edilir. Buz üzerinde yemekleri tutun. </li><li> CO 2 ile farelerin Euthanize. </li><li> Diseksiyon yüzey Güvenli fare yukarı bakacak ventral tarafta. Sterilizasyon için% 70 etanol ve kürk aşağı keçeleşmiş sağlamak için Sprey fare. </li><li> Cerrahi makas kullanarak, sadece genital Yukarıdaki karın derisinde bir ventral kesi yaparak diseksiyon başlar. Çene insizyon yukarı uzatın. </li><li> Midline itibaren, tüm uzuvları boyunca kesi uzanması. Gevşek cilt geri çekin ve diseksiyon yüzeye aşağı pin. </li><li> Timus Harvest: <ol><li> Bir kesi yapmak için sternumun alt ucu kaldırın.</li><li> Karaciğer kaçınmak, göğüs kafesi ayırmak ve sonra her tarafa göğüs kafesi kesmek için diyafram kesti. Akciğer ve kalp önlemek için dikkat çekici, her iki tarafta göğüs kafesi yukarı kesin. </li><li> Göğüs kafesi arkasında forseps ile yavaşça çekin. Timus kalp üzerinde bulunan bir beyaz, bilobe organdır. Lobların alt kavramak için forseps düz kenar kullanarak yavaşça timus çekin ve ekran örgü üzerine yerleştirin. </li></ol></li><li> Dalak Harvest: <ol><li> Dalak karaciğer aşağıda farenin karın boşluğunda, sol tarafında yer alan kırmızı, sörf tahtası şeklinde bir organdır. </li><li> Karın boşluğuna bir kesi olun. Yavaşça bağ dokusu uzakta tease ikinci çifti kullanılarak, forseps bir çift ile dalak dışarı çekin. Ayrı bir boy gözenekli elek üzerinde dalak yerleştirin. </li></ol></li></ol> 2. Hücre Hazırlanması <ol><li> 3 ml şırınga bir pistonu kullanarak, eziyetsadece bağ dokusu ve yağ kalıntıları kadar ekran örgü içine organları. Çanak HBSS üç kez örgü durulayın. Her doku örneği için yeni bir dalgıç kullanın. <ol><li> Doku homojenleştirici için diğer seçenekler bu yöntem yerine kullanılabilir. </li></ol></li><li> Hemasitometre kullanarak hücreleri saymak. </li><li> 335 santrifüje edilerek pellet hücreler 4 ° C &#39;de 5 dakika xg </li><li> Oda sıcaklığında 10 dakika için ACK lizis tamponu 500 ul (0.15 M NH4CI, 10 mM KHC0 3, 0.1 mM Na 2 EDTA, pH 7.2) içinde süspanse splenositlerin. 20 süspanse timositleri x 10 6 hücre / ml FACS tampon (PBS,% 1 FCS,% 0.02 sodyum azid) ve buz üzerinde bir kenara koyun. </li><li> HBSS 5 ml ekleyerek izotoniklik için splenositlerin dönün. Pelet 20 santrifüj ve tekrar süspansiyon tarafından splenositlerin x 10 6 hücre / FACS tampon ml. Hücrelerinin steril kalması gerekiyorsa, steril RPMI +% 10 FCS kullanabilirsiniz. </li></ol> 3.Akış sitometrisi için Boyama Hücreleri Bu protokolün amacı, yabani-tip (WT) ve HY cd4 fareler kullanarak pozitif ve negatif seçim analizi için stratejilerin altını çizmektir. Flow sitometri deneysel tasarım, satın alma, ve analizi ile ilgili genel değerlendirmeler için, biz Tung ark mükemmel bir inceleme okuyucuları bakın. 11. <ol><li> Alikot 4 x 10 96-kuyulu plakanın her bir kuyucuğa akış sitometrisi numune başına 6 timositlerde yanı sıra, telafi kontrol başına 1 x 10 6 WT splenositlerin. Transgenik fareler kullanarak, size deneyde bir denetim olarak WT fare içermelidir. </li><li> Buz üzerinde 10 dakika için anti-CD16/32 (klon 2.4G2) ile hücreler inkübe ederek Fc reseptörleri bloke ederler. </li><li> 5 dk için 4 ° C sıcaklıkta 335 x g&#39;de plaka Spin. Kapağını kaldırın ve bir kez plaka iterek kuyulardan sıvı dağıtmak, bir lavabo içine doğru bakmalıdır. FACS tamponu içinde 200 ul her bir kuyu yeniden süspanse edin. Yıkama tekrarlayın. </li><li> Aşağıda listelenen de FACS tampon başına 200 ul içinde seyreltme göre her bir antikor için optimum konsantrasyonu kullanılarak, antikor kokteyller hazırlayın. Her bir antikorun en uygun konsantrasyon pozitif ve negatif nüfusun büyük ayırma vermek için gereken en düşük konsantrasyon olarak tanımlanmıştır. , Belirli bir antikor için negatif bir nüfus var ise, bir antikor izotip dahil edilmelidir. İstenirse kuyu başına toplam hacmi (kuyu başına 100 ul) örneğin küçültülmüş olabilir. <ol><li> WT timüs: anti-TCRβ, anti-CD4, anti-CD8, anti-CD69 ya da anti-CD5, anti-CD24 </li><li> Timus HY CD4: anti-TCR HY (T3.70), anti-CD4, anti-CD8, anti-CD69 ya da anti-CD5, anti-CD24 CD69 lo ve timositleri yılında CD69 yüksek nüfus daha iyi ayrılması elde etmek için, onun tarafından izlenen bir biotinlenmiş anti-CD69 primer antikor kullanılması tavsiye edilir ikincil bir florokrom-konjuge streptavidin içeren leke. Biz uCD5 boyama için se aynı stratejiyi. </li></ol></li><li> Karanlıkta buz üzerinde 30 dakika süre ile FACS tamponu içinde antikor kokteyli 200 ul hücre ile inkübe edin. </li><li> Antikor kokteyli boyama ile eş zamanlı olarak, tek bir florokrom-konjuge antikor ile her kompanzasyon kontrolü leke. İdeal olarak, dengeleme boyama antikor kokteyli içinde kullanılan aynı antikorları içerir. Birçok antijen test ediliyor Ancak, her bir antijen için boyama büyük deneyler için uygun olmayabilir. Bu nedenle, anti-CD4, anti-CD8 ya da ya da için yüksek renklendirme, bu antijenlerin ifadesi ya da telafi boncuk kullanımına bağlı olarak tavsiye edilir. Karanlıkta buz üzerinde 30 dakika süre ile FACS tamponu içinde Leke. Bir telafi kontrolü boyanmadan bırakın. </li><li> FACS tamponu ile iki kez hücreleri yıkayın. </li><li> FACS tampon ve FACS tüplerine transfer süspanse edin hücreleri. </li><li> Akış sitometresinde örnekleri edinin. FSC-A edinimi ve FSC-W dahilçiftli ayrımcılık llow. </li></ol> 4. Akım Sitometri Veri Analizi - Sigara TCR Transgenik Fare Biz flow sitometri veri analizi için FlowJo kullanın. Non-TCR transgenik fareler için yolluk strateji 2A Şekil bakın. <ol><li> SSC, &quot;lenfosit&quot; nüfus üzerindeki elektronik kapıdan FSC-A kullanma. </li><li> &quot;Lenfosit&quot; nüfus içinde, hücre çiftler ve aggregrates dışlamak için elektronik kapı FSC-W lo nüfus FSC-W tarafından FSC-A kullanın. Bu &quot;singlet&quot; kapısıdır. </li><li> &quot;Singlet&quot; kapısı, CD4 arsa CD8 olayları kullanma. DN, DP donuk, DP aydınlık için geçitler çizin, CD4 Şekil 2B&#39;de gösterildiği gibi + CD8 lo, CD4SP ve CD8SP nüfus. </li><li> CD4SP ve CD8SP kapılar altında, CD24 araziler tarafından TCRβ oluşturun. Şekil 2C tasvir gibi kapıları çizmek için kadranda gating aracını kullanın. </li><li> A ne oluşturmaCD69 ile TCRβ: &quot;tekli&quot; kapısı w arsa. Kapıları A, B, C, D, Şekil 2B&#39;de gösterilmiştir. Çizme </li><li> CD5 tarafından TCRβ: &quot;tekli&quot; kapıdan başka bir arsa oluşturun. Şekil 2E tasvir gibi kapıları i, ii, iii, iv çizin. Bu pozitif seleksiyon incelenmesi için başka bir stratejidir. </li><li> Kapıları i, ii, iii, iv, A, B, C, D (Şekil 2B, E) &quot;singlet&quot; altından DN, DP donuk, DP parlak, CD4 int, CD4SP ve CD8SP kapıları uygulayın. </li><li> Eğer hedef nüfus ulaşana kadar sonraki her kapının frekansını organ selülarite çarparak belirli bir alt kümesi hücrelerin sayısını hesaplayın. Örneğin, TCRβ hi CD69-CD8SP timositlerin sayı timüs sellüler x% TCR BHI tarafından belirlenecektir CD69-(D fraksiyonu) x% CD8SP. <ol><li> Sayma zaten canlı ve ölü hücreleri böylece &quot;lymphoc içermez dikkate alırhesaplamalarda YTE &quot;kapısı. </li></ol></li></ol> 5. Akım Sitometri Veri Analizi - TCR Transgenik Fareler TCR transgenik fareler için yolluk strateji 3A Şekil bakın. <ol><li> Adımlarda, önceki bölümlerde olduğu gibi 4.1 ve 4.2 izleyin. </li><li> &quot;Singlet&quot; kapı altında T3.70 üzerine SSC arsa ve kapı bir T3.70 oluşturmak + hücre popülasyonu antijen-spesifik T hücreleri (Şekil 3B) analiz etmek. Bazı durumlarda, bu non-spesifik antijen (T3.70-) T hücre popülasyonu bu nüfusu karşılaştırmak için ilgi çekici olabilir. </li><li> Içinde T3.70 + nüfus, beraberlik DN, DP, CD4SP ve CD8SP kapıları (Şekil 3C). <ol><li> Çok az T3.70 + hücreleri olacak gibi WT kontrol örnekleri için, &quot;tekli&quot; kapısının altına bu kapılardan çizmek veya bir T3.70 kapısı oluşturun. </li></ol></li><li> CD8SP kapısı altında, oluşturmakCD24 arsa T3.70. Dört nüfus (Şekil 3F) içine hücre bölmek için kadranda gating aracını kullanın. </li><li> WT fareler ve HY cd4 farelerin T3.70 + DP bölmesi (Şekil 4A) DP bölmesinde CD69 ekspresyonu gösteren Kalıplı bir histogram oluşturun. CD5 ekspresyonu (Şekil 4B) incelemek için tekrarlayın. </li><li> 4.8 gibi ilgi her alt hücrelerin mutlak sayısını hesaplayın. </li></ol>

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K., Cado, D., Winoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+orphan+steroid+receptor+Nur77+in+apoptosis+accompanying+antigen-induced+negative+selection.">A role for the orphan steroid receptor Nur77 in apoptosis accompanying antigen-induced negative selection.</a> Immunity. 3, 273-282 (1995).</li><li>Zhou, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Nur77/Nurr1+leads+to+inefficient+clonal+deletion+of+self-reactive+T+cells.">Inhibition of Nur77/Nurr1 leads to inefficient clonal deletion of self-reactive T cells.</a> J. Exp. Med. 183, 1879-1892 (1996).</li><li>Baldwin, T. A., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+analysis+of+clonal+deletion+in+vivo.">Transcriptional analysis of clonal deletion in vivo.</a> J. Immunol. 179, 837-844 (2007).</li></ol>

Çıkar çatışması ilan etti.

Burada sunulan protokol olmayan TCR transjenik ve TCR transgenic farelerde pozitif ve negatif seçim incelemek için kullanılabilir. Bu protokol, yüzey antijenlerinin boyama açıklanmaktadır. Moleküler mekanizmalarının daha fazla analiz için, genellikle hücre içi boyama yapmak için gereklidir. Biz transkripsiyon faktörleri için en intraselüler proteinler ve BD Biosciences Foxp3 Boyama Seti BD Biosciences Cytofix / Cytoperm Kiti kullanın. Biz genellikle hemen boyama sonra örnekleri kazanır. Bununla birli...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> HyClone Hank&#39;in dengeli tuz çözeltisi </td><td> Thermo Scientific </td><td> SH30030.02 </td><td></td></tr><tr><td> Metal örgü ekranlar </td><td> Cedarlane </td><td> CX-0080-E-01 </td><td></td></tr><tr><td> Petri kapları (60 x 15 mm) </td><td> Fisher Scientific </td><td> 877221 </td><td></td></tr><tr><td> Enjektörler (3 ml) </td><td> BD Biosciences </td><td> 309657 </td><td></td></tr><tr><td> Konik boru (15 ml) </td><td> Sarstedt </td><td> 62.554.205 </td><td></td></tr><tr><td> Mikroskop </td><td> Zeiss - Primo Yıldız </td><td> 415500-00XX-000 </td><td></td></tr><tr><td> Hemasitometre </td><td> HausserBilimsel </td><td> 3110 </td><td></td></tr><tr><td> 96-kuyulu plakalı </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> Kanallı pipet </td><td> Fisherbrand </td><td> 21-377-829 </td><td></td></tr><tr><td> Fetüs buzağı serumu </td><td> PAA </td><td> A15-701 </td><td></td></tr><tr><td> Fosfat tamponlu salin </td><td> Fisher Scientific </td><td> SH3025802 </td><td></td></tr><tr><td> Sodyum azid </td><td> BT Baker Chemical Co </td><td> V015-05 </td><td></td></tr><tr><td> FCR engelleme reaktif </td><td></td><td></td><td> Klon 2.4G2 </td></tr><tr><td> Anti-fare HY TCR </td><td> eBioscience </td><td> XX-9930-YY * </td><td> Klon T3.70 </td></tr><tr><td> Anti-fare CD4 </td><td> eBioscience </td><td> XX-0042-YY * </td><td> Klon RM4-5 </td></tr><tr><td> AYüzgeç-fare CD8α </td><td> eBioscience </td><td> XX-0081-YY * </td><td> Klon 53-6,7 </td></tr><tr><td> Anti-fare CD24 </td><td> eBioscience </td><td> XX-0242-YY * </td><td> Klon M1/69 </td></tr><tr><td> Anti-fare TCRβ </td><td> eBioscience </td><td> XX-5961-YY * </td><td> Klon H57-597 </td></tr><tr><td> Anti-fare CD69 biotinlenmiş </td><td> eBioscience </td><td> 13-0691-YY * </td><td> Klon H1.2F3 </td></tr><tr><td> Anti-fare CD5 biotinlenmiş </td><td> eBioscience </td><td> 13-0051-YY * </td><td> Klon 53-7,3 </td></tr><tr><td> Streptavidin </td><td> eBioscience </td><td> XX-4217-YY * </td><td></td></tr><tr><td> Akış sitometresi </td><td> BD Biosciences - FACS Canto </td><td> 338962 </td><td></td></tr><tr><td> FACS tüpler </td><td> BD Biosciences </td><td> 352052 </td><td></td></tr><tr><td> Flow sitometri analiz yazılımı </td><td> TreeStar - Flowjo </td><td> FlowJo v7 / 9 </td><td></td></tr><tr><td> HyClone RPMI - 1640 orta </td><td> Thermo Scientific </td><td> SH30027.01 </td><td></td></tr><tr><td></td><td></td><td></td><td> Florokrom ve YY Şişe boyutuna göre değişir tarafından * XX değişir. </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

Sağlıklı bir bağışıklık sistemi kendi antijenlerine tolerans kalırken T hücrelerinin yabancı antijenleri yanıt gerektirir. T hücre reseptör (TCR) α ve β lokusların Rastgele düzenlenmesi öz ve yabancı hem antijen özgüllüğü engin çeşitliliği ile T hücre repertuarı oluşturur. Timusta gelişimi sırasında repertuar seçimi güvenli ve kullanışlı T hücreleri üretmek için önemlidir. Timik seçiminde Kusur otoimmün ve immün bozuklukları 1-4 gelişmesine katkıda bulunur. T hücre progenitörler CD4 veya CD8 co-reseptörleri ifade etmezler çift negatif (DN) timositleri olarak timus girin. ΑβTCR hem eş-reseptör ekspresyonu çift pozitif (DP) aşamada gerçekleşir. Öz-peptid-MHC (pMHC) timus hücreleri tarafından sunulan ile αβTCR Etkileşimi DP timosit kaderini belirler. Yüksek afinite etkileşimleri olumsuz seçimi ve ortadan kalk yolkendinden reaktif timositlerin tion. CD4 veya kendinden MHC 5 tarafından sunulan yabancı antijenleri tanıma yeteneğine CD8 tek pozitif (SP) T hücrelerinin pozitif seçimi ve gelişimi Düşük afinite etkileşimleri sonucu. Pozitif Seçimi olgun T hücrelerinin üretimi gözlemleyerek bir poliklonal (yabani-tip) TCR repertuarı ile farelerde incelenebilir. Bununla birlikte, küçük antijen-spesifik popülasyonlarının silme içerir negatif seçim çalışma için ideal değildir. Birçok model sistemler negatif seçim çalışma ama fizyolojik olaylar 6 özetlemek için yeteneklerini değiştirmek için kullanılan edilmiştir. Eksojen antijen uygulamasından timositlerde 7-9 non-spesifik silme yol açabilir Örneğin, timositlerde in vitro uyarımı olarak, iç içe işlemine dahil olan timik ortam yoksundur. Şu anda, in vivo negatif seleksiyon eğitim için en iyi araçları bir transgresyon ifade fareler vardırself-antijen endojen için ENIC TCR özgü. Bununla birlikte, birçok klasik TCR transjenik modelleri prematüre negatif seçim ile sonuçlanan, DN aşamada transgenik TCRα zincirinin erken ifade ile karakterize edilir. Bizim laboratuvar olarak 10 yabani-tip farelerden oluşan negatif seleksiyon SP geçiş DP sırasında meydana sağlayan, transgenik HY TCRα şartlı DP aşamada ifade edildiği HY cd4 model geliştirdi. Burada, HY cd4 fare modelinde timik pozitif ve negatif seçim incelemek için bir akış sitometri tabanlı protokol açıklar. HY CD4 farelerde, negatif seleksiyon çok fizyolojik olmakla birlikte, bu yöntemlerin diğer TCR transjenik modelleri ile de uygulanabilir. Biz de herhangi bir genetik manipüle fare için geçerli bir poliklonal repertuar olumlu seçimi analiz için genel stratejiler sunacak.

Fizyolojik TCR transgenik modeller ve WT farelerde, pozitif seleksiyon antijen karşılaşma sonrasında DP donuk aşamaya geçmeden önce DP parlak aşamada başlar. DP donuk timositleri ardından CD4SP veya CD8SP timositleri (Şekil 2B) olmadan önce bir geçiş CD4 + CD8 lo sahne girin. Olgun SP timositleri yüksek TCR ekspresyonu ve CD24 (Şekil 2C) kaybı ile karakterize edilir. Profil CD69 veya CD5 tarafından TCRβ inceleyere...

Watch this Scientific Journal Video about Examination of Thymic Positive and Negative Selection by Flow Cytometry at JoVE.com

Examination of Thymic Positive and Negative Selection by Flow Cytometry

Biz T hücre gelişimini incelemek üzere bir akım sitometri tabanlı bir yöntem sunmak<em&gt; In vivo</em&gt; Bir yabani-tip veya T hücre reseptörü transgenik arka plan üzerinde genetik manipüle fare kullanarak.

Akım Sitometri tarafından Timik Pozitif ve Negatif Seçim incelenmesi

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

examination-thymic-positive-negative-selection-flow

Research

JoVE Journal

Immunology and Infection

21.6K Görüntüleme.  University of Alberta. Biz T hücre gelişimini incelemek üzere bir akım sitometri tabanlı bir yöntem sunmak In vivo Bir yabani-tip veya T hücre reseptörü transgenik arka plan üzerinde genetik manipüle fare kullanarak.

Video: Examination of Thymic Positive and Negative Selection by Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Sabit ve Yaşam Hücreler Sitometrisi ile Calpain Aktivitesi Ölçme

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

İmmünoloji ve Enfeksiyon

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Insan In Vitro Bağışıklık Dengesizlik Erken Devlet Tanıma Tarama Aracı Olarak Bastırılması

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Sitometrisi ile Primer İmmün hücre alt Floresan Nanoparçacık Etkileşimleri Algılama

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

Akış Cytometry tarafından Kemirgen Plasmodium parazitemi Vivo Değerlendirilmesi ve Merozoit Invasion

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Akış Sitometrisi ile Sıçan Böbrek gelen Makrofagların Fenotipik Karakterizasyonu

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fare kemik iliğinden plazmasitoid dendritik hücreleri Saflaştırılması floresans ile aktive edilen hücre sınıflandırma

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Yalıtım ve fare ve insan alveoler makrofajlar Vitro kültürü

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

İnsan monosit alt kümeleri tam kan tarafından karakterizasyonu Akış Sitometresi Analizi

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

İki-fluorochrome tarafından yedi bağışıklık hücre alt kümeleri ayrımcılık Akış Sitometresi

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Nötrofil kütle sitometri analizi için tüm kemik Iliği hazırlanması-Lineage hücreler

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...