Steve McBryant ve Jeff Hansen Biz fare kontrolü ile ilgili yardım için Ultrasantrifügasyon ve Patti Kiser yardım için teşekkür ederim. Ulusal Sağlık Enstitüleri Sinir Hastalıkları ve İnme Ulusal Enstitüsü, hibe 5R01NS056379-02 bu işi finanse edilmektedir.

<html> 1. Prion Çubuklar Arındırıcı ve Etiketleme Bu protokol daha önce yayınlanmış 18 uyarlanmıştır <ol><li> Ticari bir blender maksimum hızda 1 dakika, 2 dakika sonra buz üzerinde buz gibi homojenleştirici tampon 900 mL prion ile enfekte beyin dokusu 100 gram (HB, 320 mM sakkaroz, 150 mM NaCl ve 4mm EDTA içeren 1X PBS) karıştırılır. Üç kez tekrarlayın. </li><li> 3000 xg&#39;de 10 dakika ve 4 ° C Homojenat Santrifüj Çıkarın ve buz üzerinde supernatant kaydetmek. 1L HB pelet yeniden askıya alma ve 1.1 ve 1.2 adımları tekrarlayın. </li><li> 100.000 xg, 0 ° C 60 dakika Havuzu süpernatantlar ve santrifüj Süpernatant atın. </li><li> Yeniden askıya 100 ml 1X Tris pelet tamponlu tuz (10 mM Tris-HCl, 0.1 mM NaCl ve 1 mM EDTA) içeren% 2 Triton X-100 (TBST). Tahmin Bradford veya benzer tahlil protein konsantrasyonu ve 5 mg / ml TBST ayarlayabilirsiniz. 30 dakika buz üzerinde soğutun. </li><li> 100.000 xg, 0 ° C 30 dakika santrifüj Süpernatant atın ve 50 ml TBST sonra 100 mL TBS ile yıkayın pelet. 100 ml 1X PBS% 1 sarcosyl ve proteaz inhibitörü kokteyl içeren pelet Re-37 az 120 dakika süreyle askıya alma ve heyecan ° C. </li><li> 100.000 xg, 10 ° C, 60 dakika için 320 mM sukroz ve santrifüj 900 mL Katman örnek Süpernatant atın ve 10 ml 2,3 M NaCl içinde yeniden askıya pelet,% 5 sarcosyl. </li><li> Örnek 5 x 40 saniye, 1 dakika aralıklarla buz üzerinde% 70 maksimum güç sonikasyon. </li><li> 13.000 xg, 4oC az on beş dakika için Eppendorf tüpleri ve santrifüj içine örnek kısım 1 ml. TBS ve mağaza ile iki kez yıkayın pelet @ -70 ° C veya adım 1.9 florokrom ile konjuge. </li><li> Prion çubuklar kullanarak DyLight 649 1 şişe saf Konjugat 1 tüp, üretici firmanın protokolüne göre florokrom. </li><li> PBS için filtre sütunları ile diyaliz veya santrifüj Exchange DyLight etiketleme tampon. -70 ° C kullanıma hazır olana kadar uzakta ışıktan 20 mcL alikotları saklayın. </li></ol> 2. Prion Aşılama ve Hücre Kurtarma Bu bölümde, intraperitoneal prion aşılama ve periton, mediastinal ve mezenterik lenf düğümleri ve dalak hücreleri kurtarma kapsar. <ol><li> Kullanımdan hemen önce floresan prion çubuklar PBS ile 1:50 sulandırınız. Başparmak ve işaret parmağı ile dorsal boyun kürk ense fare, hafifçe yüz ve serçe parmak ile kuyruk dizginlemek için fare açmak. Başının üstünde arka gövde yükselten ve onu biraz baş aşağı tutarak, fare Supinate. </li><li> 30G insülin şırınga kullanarak, sakroiliak eklem anterior orta hat ve 1-2 cm 1 cm lateral floresan prion çubuklar 100 mcL enjekte edilir. 1 cm deri yoluyla mediale 45 ° yönetti iğne takın. Kontrol grubu olarak floresan bilyeler veya PBS tek başına 1:50 seyreltme kullanın. Analizden önce ayrılan zaman fareler inkübe edin. </li><li> Onaylanmış hayvan bakımı göre fareler Euthanize ve komite protokollerini kullanmak. PBS 10 ml, 12 ml şırınga ve 25G iğne kullanarak periton boşluğuna enjekte edilir. Bir dakika boyunca hafifçe boyuna karkas sallayın. Karkas diseksiyonu devam ederken, buz üzerinde, 15 ml konik tüp ve yedek aynı şırınga ve 16G iğne kullanarak periton hücre süspansiyonu kurtarın. </li><li> Karkas ventral tarafında% 70 etanol ile iyice ıslatın. Sadece forseps ile göğüs kafesi altındaki orta hat cilt kaldırın ve makas ile deri yoluyla küçük bir kesi kesmek. Her iki ucunda iki orta hatta 2 cm kesiler yanal, daha sonra baş ve kuyruk her iki yönde de deri yoluyla 3-5 cm uzunluğunda bir kesi yapmak, orada başlayarak. Geri Peel ve peritoneal ve torasik boşlukları ortaya çıkarmak için cildi pin. Dikkatle periton çevreleyen ince saydam membranlar kesti ve mediastende ortaya çıkarmak için toraks yansıtacak. </li><li> Trakea ventral kenarında brakiyosefalik arterin medial bitişik timus biraz dorsal ve yanal bitişik 2-4 mediastinal lenf düğümleri,, bulun ve çıkarın. Bulun ve yumuşak beyaz mezenterik dokusunda gömülü altı mezenterik lenf düğümleri, yukarı kaldırmak posterior abdominal duvar çapa bağırsak. Dalak, sadece orta hat ve lateral dorsal karnın sol tarafında bağırsaklarında bulunan uzun, koyu kırmızı organ bulun ve çıkarın. Rezerv lenf düğümleri 1 ml RPMI 1640 orta buz üzerinde diseksiyon tamamlanana kadar. </li><li> 1 ml şırınganın pistonu kullanarak bir plastik Petri kabındaki RPMI 3 mL 40 mikron mesh süzgeç ile ıslatarak yumuşatmak ve lenf düğümleri basın. RPMI ek bir 2 mL süzgeç durulayın ve 15 ml konik bir tüpe 5 ml hücre süspansiyonu transfer. Petri kabı RPMI ek bir 5 ml ile durulayın ve aynı tüp transferi. 250 xg&#39;de 5 dakika santrifüj, 1 ml FACS tampon (1X PBS,% 1 sığır serum albumin ve 10 mM EDTA) supernatant, yeniden askıya hücre pelletini atmak ve 1.5 ml Eppendorf tüp transferi. </li></ol><html> ove_title &quot;&gt; 3 Hücre Boyama ve Sitometrisi Iyi karakterize bağışıklık hücre yüzey belirteçleri karşı floresan antikor kullanarak bağışıklık hücre alt grupları tanımlamak için aşağıdaki tipik bir boyama protokoldür. Tüm antikorlar kullanmadan önce hemen FACS tampon içine seyreltilmiş. <ol><li> Eppendorf tüpleri içine hücreleri ve kısım 10 6 hücre güvenin. Santrifüj hücreleri (2.7) ve 1 ml FACS tamponu ile yıkama pelet 2X. </li><li> Endojen Fc reseptörlerini bloke 2 ng / ml sıçan anti-fare CD16/32 100 mcL hücreleri buz üzerinde 20 dk inkübe edin. Pelet ve FACS tamponu ile yıkama hücreleri. </li><li> Bir saat boyunca her bir örnek ve buz üzerinde inkübe 1ng / ml uygun floresan antikorlarının (y) ler 100 mcL ekleyin. Kontrol hücreleri tek başına FACS tampon 100 mcL ekleyin. Pelet ve iki kez yıkayın hücreleri FACS tampon. </li><li> 1 ml ACK tamponu (NH 4 Cl 150 mM, 1 mM KHCO 3 ve 0.1 mM EDTA), kırmızı kan hücreleri lyse için 1 dakika boyunca buz üzerinde inkübe hücreleri yeniden askıya . Pelet ve FACS tamponu ile yıkama hücreleri. 1 ml FACS tampon hücreleri yeniden askıya. </li><li> Genellikle 405, 488 ve 633 nm eksitasyon lazerler ve 09:55 floresans emisyon dedektörleri, çok renkli algılama ve analiz yeteneğine sahip bir akış sitometresinin kullanarak hücrelerin veri toplama. </li></ol> 4. Temsilcisi Sonuçlar: Biz ham prion çubuklar saflaştırılmış, E2 geyik beyin Homojenat (hat 1 ve 2) prion ile enfekte beyin Homojenat (Şekil 1) Deterjan çözünürleştirme ve Ultrasantrifügasyon proteaz K prion çubuklar (1 ve 3 şeritli karşılaştırmak), büyük ölçüde zenginleştirilmiş dirençli (4 şeritli). İlginçtir ki, sindirilmemiş saflaştırılmış prion çubuklar prionlar için dramatik zenginleştirme gösteren çarpıcı bir şekilde sindirilir E2 beyin Homojenat benzer bir glycoform profili görüntülenir. Aynı tedavi normal beyin Homojenat hiçbir PK-dirençli PrP C (şerit 5 ve 6) üretti. Belirli hücre popülasyonlarının Flow sitometrik analizi antijen iki saat periton hücreleri ev sunulması ve floresan boncuk ve prion çubuklar, PBS enjekte kontroller üzerinde dramatik bir artış floresan (Şekil 2) ile kanıtlandığı gibi korumak göstermektedir. Boncuk (Şekil 2H-N) hücreleri ve prion (Şekil 2O-U) inokule önemli floresan görüntülenir fareler ise, arka plan yukarıda floresan (Şekil 2A-G) görüntülenen PBS tedavi kontrolleri periton hasat hücre yok, özellikle monositler (Şekil 2I ve P), dendritik hücreler (Şekil 2K ve R) ve makrofajlar (Şekil 2L ve S) ve bazı Nötrofiller (Şekil 2J ve Q) ve daha az B hücreleri (Şekil 2T). Boncuk ve Prion taşıyan monositler, dendritik hücreler ve makrofajlar da mediastinal lenf düğümleri aşılama (Şekil 2AD ve AK, AF ve AM ve AG ve AN, sırasıyla) ise az ya da hiç PrP CWD taşıyan Nötrofiller iki saat sonra bulundu , B veya T hücreleri (Şekil 2AE ve AL, AH ve AG ve sırasıyla AI ve AP) tespit edilmiştir. Biz herhangi bir boncuk veya prion çubuklar dalak veya mezenterik lenf düğümleri (veriler gösterilmemiştir) tespit edildi. Toto, bu veriler, bağışıklık hücrelerini trafik diğer partikül antijenleri çok benzer prion göstermektedir. <table border="1"><tbody><tr><td> REAKTİF </td><td> Florokrom </td><td> İKAZ LAZER (nm) </td><td> PEAK EMİSYON (nm) </td></tr><tr><td> Prion çubuklar </td><td> DyLight 649 </td><td> 633 </td><td> 674 </td></tr><tr><td> 1 mikron boncuk </td><td> AlexaFluor 660 </td><td> 633 </td><td> 685 </td></tr><tr><td> Antikorlar </td><td></td><td></td><td></td></tr><tr><td> CD11b </td><td> eFluor 450 </td><td> 405 </td><td> 450 </td></tr><tr><td></td><td> Fikoeritrin-Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> CD11c </td><td> R-Fikoeritrin </td><td> 488 </td><td> 575 </td></tr><tr><td></td><td> Fikoeritrin-Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> Ly6C </td><td> Fluoroisothiocyante </td><td> 488 </td><td> 518 </td></tr><tr><td> Ly6G </td><td> R-Fikoeritrin </td><td> 488 </td><td> 575 </td></tr><tr><td> CD21 </td><td> DyLight 488 </td><td> 488 </td><td> 518 </td></tr><tr><td> B220 </td><td> Allophycocyanin-Cy7 </td><td> 633 </td><td> 785 </td></tr><tr><td> CD3 </td><td> R-Fikoeritrin </td><td> 488 </td><td> 575 </td></tr></tbody></table>&quot;&gt; Tablo 1 floresan reaktiflerin spektral özellikleri <img src="/files/ftp_upload/2349/2349fig1.jpg" alt="Şekil 1" /> Şekil 1. Enfekte beyin Homojenat prion çubuklar Arıtma Biz toplu prion çubuklar (şerit 3 ve 4) saflaştırılmış hangi malzeme başlangıç ​​olarak bir CWD enfekte geyik (E2, şerit 1 ve 2)% 10 ham beyin Homojenat kullandı. Normal beyin Homojenat (şerit 5 ve 6) bulunmamış iken, ham ve arıtılmış malzemeleri proteaz K tedavisi (4 hat 1) için karakteristik bir direnç gösterdi. Molekül ağırlığı işaretleri leke sol Kd gösterilmiştir. BH, beyin Homojenat. <img src="/files/ftp_upload/2349/2349fig2.jpg" alt="Şekil 2" /> Şekil 2. Aşılama (panelleri AG ve V-AB), floresan boncuk (HN ve AC-AI) veya Prion çubuklar (OU ve AJ-AP) PBS iki saat sonra periton mediastinal lenf düğümleri bağışıklık hücreleri ticareti prionlar Akış sitometrik analizi peritoneal lavaj sıvısı (AU) ya da iki saat sonra mediastinal lenf düğümleri (V-AP) hasat fareler ve hücrelerin periton içine enjekte edilir. PBS (paneller A ve V), floresan boncuk (panel H ve AC) ve prion çubuklar (paneller O ve AJ) ile tedavi edilen farelerden alınan ilk sütunda göstermek hücrelerinde Grafikler. Floresan hücreleri (kırmızı veya yeşil noktalar) ve toplam hücreleri (siyah nokta) göreceli büyüklüğü (forward scatter), taneciklik (yan dağılım) ve floresan toplam canlı hücreler oranı göstermek için çizilir. Granülosit karakteristik hücrelerin önemli sayıda floresan boncuk ve çubuklar korudu. Hücreler aynı zamanda bağışıklık hücre yüzey işaretleyicileri ve LYG-Ly6C + monositler için kapı (panel B, I, P, W, AD ve AK), Ly6c-CD11c-CD11b + Ly6G + nötrofil (C, J, Q, X, AE karşı antikor ile boyandı edildi ve AL), Ly6G-Ly6C-CD11b + CD11c + dendritik hücreler (D, K, R, Y, AF ve AM), Ly6G-Ly6C-CD11c-CD11b + makrofajlar, (E, L, S, Z, AG ve AN), CD3 -B220 + CD21 + B hücreleri (F, M, T, AA, AH ve AO) ve CD21-B220-CD3 + T hücreleri (G, N, U, AB, AI ve AP). Monositler, dendritik hücreler ve makrofajlar floresan boncuk ve prion çubuklar korudu periton boşluğuna (panelleri I ve P, K ve R ve L, G, sırasıyla) ve mediastinal lenf düğümleri (paneller AD ve AK, AF ve AM ve AG taşınan sırasıyla BİR), bu iki parçacık benzer alımı ve ticareti gösteren. Nötrofiller boncuk (J) ve daha az çubukları (S) önemli bir miktarı korunur, ancak lenf düğümleri (AE ve AL) teslim etmek üzere başarısız oldu. B hücreleri ve taşınan bazı çubuklar (T ve AO) ama hemen hemen hiç boncuk (M ve AH) korudu. T hücreleri boncuk ne çubuklar (N, U, AI ve AP) ne kaçırılmaktadır.

<ol>
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Burada periferik prion enfeksiyonu erken olayları büyük ölçüde takibini kolaylaştıran in vivo etiketleme ve izleme prionlar için bir protokol göstermektedir . Bu protokol, net bir şekilde endojen PrP C ayırt etmek için, ön-etiketleme zenginleştirilmiş prion inokulum, in vitro 9 ve in vivo 3,12 izleme prion alımını geçmişte girişimleri büyük ölçüde geliştirir. Çünkü bulaşıcı prionlar ve PrP C aynı birincil amino asit dizilimi...

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<td>CWD-infected elk brain</td>
<td>&nbsp;</td>
<td>Private elk farm in Colorado</td>
<td>&nbsp;</td>
<td>Use any non-human prion-infected brain</td>
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<td>Blender</td>
<td>&nbsp;</td>
<td>Oster</td>
<td>6694-015</td>
<td>Use any commercial blender</td>
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<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>SS34 rotor</td>
<td>Use any centrifuge /rotor that can reach 3000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>50.2 Ti rotor</td>
<td>Use any ultracentrifuge /rotor that can reach 100,000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Bradford Reagent</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>B6916</td>
<td>&nbsp;</td>
<tr>
<td>Complete mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11 836 170 001</td>
<td>&nbsp;</td>
<tr>
<td>Sonicator</td>
<td>&nbsp;</td>
<td>Misonix</td>
<td>MP4000X</td>
<td>Use any horn or probe sonicator set to ~70% max power</td>
</tr>
<tr>
<td>DyLight antibody Labeling kit</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>53050</td>
<td>&nbsp;</td>
<tr>
<td>microcentrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>55430R</td>
<td>Use any refrigerated microcentrifuge that can achieve 13,000x g</td>
</tr>
<tr>
<td>centrifugal filter columns</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>Microcon YM-100</td>
<td>Use any filter or dialysis membrane with 100 Kd molecular weight cutoff</td>
</tr>
<tr>
<td>8-40 week-old FVB mice</td>
<td>&nbsp;</td>
<td>Charles River</td>
<td>207</td>
<td>Use any inbred mouse strain</td>
</tr>
<tr>
<td>1 &mu;m red fluorescent beads</td>
<td>&nbsp;</td>
<td>Phosphorex</td>
<td>2307</td>
<td>Use any fluorescent bead &le; 10 &mu;m</td>
</tr>
<tr>
<td>RPMI 1640 medium </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11875-093</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>fluorescent antibodies</td>
<td>&nbsp;</td>
<td>BD pharmingen</td>
<td>Various</td>
<td>Use any fluorescent antibody appropriate for your application.</td>
</tr>
<tr>
<td>flow cytometer</td>
<td>&nbsp;</td>
<td>Dakocytomation</td>
<td>CyanADP</td>
<td>Use any flow cytometer capable of multicolor fluorescence detection</td>
</tr>		</tbody>
		</table>
		</div>

monitoring immune cells trafficking fluorescent prion rods hours after intraperitoneal infection

Anormal bir formu varlığı, proteaz, dayanıklı, patolojik ve bulaşıcı bir host-kodlanmış prion proteini (PrPC) Kronik Wasting koyun cervids ve scrapie hastalığı (CWD) olarak prion hastalıkları karakterize etmektedir. Prion hipotez bu anormal conformer çoğu veya tüm bulaşıcı prion teşkil ettiğini ileri sürmektedir. Periferik prion patogenezinde erken olaylar bağışıklık sisteminin rolü ikna edici CWD ve scrapie 1-3 kanıtlanmıştır. Farelerde Transgenik ve farmakolojik çalışmalar sonrası erken dönemde enfeksiyon 4-6 prionlar istinat ve kopyalayan Kompleman sisteminin önemli bir rolü ortaya koymuştur. 7-10 dendritik hücreler tarafından prion tutma gözlemledik in vitro ve in vivo çalışmalar, insan ticareti rollerini olmaya devam etmekle beraber, 11-16 belirsiz. Makrofajlar benzer erken prion patogenezinde suçlanmıştır, ancak bu çalışmalar, enfeksiyon 3,11,17 hafta sonra meydana gelen olaylar üzerine odaklanmıştır . Bu ve önceki çalışmalarda da endojen PrP C ve bulaşıcı prionlar arasındaki farklılıklarda sorun muzdarip. İşte biz orada alınan bağışıklık hücreleri tarafından aşılama sitesinden prion alımı ve ticareti değerlendirmek için bir semikantitatif, tarafsız bir yaklaşım açıklar. Toplulaştırılmış prion çubuklar olmayan toplanan proteinleri ve sakaroz yastık üzerinden Ultrasantrifügasyon deterjan çözünürleştirme ile enfekte beyin Homojenat arınmış. Poliakrilamid jel elektroforezi, Coomassie mavi boyama ve zenginleştirilmiş pelet fraksiyonu prion çubuklar batı blotting teyit kurtarma. Prion çubuklar farelere florokrom-etiketli enjekte intraperitoneal. İki saat sonra, peritoneal lavaj sıvısı, dalak ve mediastinal ve mezenterik lenf düğümleri bağışıklık hücreleri prion çubuk tutma ve monositler, nötrofil, dendritik hücreler, makrofajlar ve B ve T hücreleri için işaretleri kullanarak çok renkli akım sitometri ile tespit edilen hücre alt grupları ölçüldü. Bu testte, enfeksiyondan sonra saat içinde bağışıklık elde hücreleri ve in vivo kaçakçılığı prionlar ilk kez doğrudan izleme sağlar . Bu testte de zor ve diğer test sistemlerinde veri yorumlama sorunları neden olabilir normalde ana hücreleri üzerinde ifade PrPC bulaşıcı, toplu prion, açıkça ayırır. Bu protokol, diğer aşılama yolları (oral, intravenöz, intranervous ve derialtı, örneğin) ve aynı zamanda antijenler (konjuge boncuk, viral, bakteriyel ve paraziter patojenler ve proteinler, yumurta) adapte edilebilir.

Watch this Scientific Journal Video about Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection at JoVE.com

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Burada hemen arındırıcı intraperitoneal aşılama ve floresan sonra enjeksiyon yerinde alımı ve hareket izleme ve bu olaylara aracılık eden hücreler karakterize enfekte beyin malzeme toplanan prion çubuklar etiketleme prion alımı ve ticareti bağışıklık hücreleri tarafından izlenmesi için yeni bir tahlil açıklar.

İntraperitoneal Enfeksiyon sonra Bağışıklık Hücreleri Ticareti Floresan Prion Çubuklar Saat İzlenmesi

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

monitoring-immune-cells-trafficking-fluorescent-prion-rods-hours

Research

JoVE Journal

Immunology and Infection

11.4K Görüntüleme.  Colorado State University. Burada hemen arındırıcı intraperitoneal aşılama ve floresan sonra enjeksiyon yerinde alımı ve hareket izleme ve bu olaylara aracılık eden hücreler karakterize enfekte beyin malzeme toplanan prion çubuklar etiketleme prion alımı ve ticareti bağışıklık hücreleri tarafından izlenmesi için yeni bir tahlil açıklar.

Video: Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

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

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hücreler stellat Yedi Adımda

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Bağışıklık Hücreleri murin aorta içinde Sitometrisi Analizi

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Fonksiyonel Kardiyak Bağışıklık Hücreleri İzolasyon

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Hücre-kaderi ardından<em&gt; E. coli</em&gt; Faj Lambda tarafından Enfeksiyon Sonrası

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Fonksiyonel ve Moleküler Çalışmalar Birincil murin Tip II alveol epitel hücrelerinde akım sitometri İzolasyonu

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Yağ Doku Bağışıklık Hücre İzolasyonu

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

Lenf düğümü Stromal Hücre Menşe Çalışmaları Lenf-yağ Pad Kimeralar Üretimi

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

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 ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

İnsan Periferik Kan immünfenotipik Analizi Antikor Kokteyller Hazırlama A Yarı-otomatik Yaklaşım

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...