Yazarlar mikroskobu deneyleri ile mükemmel teknik yardım için Glen Slocum (Wisconsin Tıp Koleji) ve Colleen A. Lavin (Nikon Instruments, Inc.) teşekkür etmek istiyorum. Gregory Blass yazının kritik redaksiyon için kabul edilmektedir. Bu araştırma (AS) 1-15-BS-172, ve (DVI) Nefroloji American Society Ben J. Lipps Araştırma Bursu hibe Sağlık hibe HL108880 ve American Diabetes Association Ulusal Sağlık Enstitüleri tarafından desteklenmiştir.

Hayvan kullanımı ve refah Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından incelenen ve onaylanan protokoller aşağıdaki Laboratuvar Hayvanları Bakımı ve Kullanımı NIH Kılavuzu&#39;na uygun olmalıdır. 1. Böbrek Gömme <ol><li> (Ancak farklı yaş ve cinsiyet diğer suşları uygun değişikliklerle kullanılabilir, bir Sprague Dawley suşu olan önerilen) 8 ila 12 haftalık erkek sıçan kullanın. </li><li> IACUC protokolü tarafından izin verilen prosedüre göre hayvan anestezisi; anestezi derinliği izlemek ve hayvan kontrol edin. cerrahi ayrıntılı bir açıklama 1.3 gerçekleştirilebilir göre - 1.8 Ilatovskaya ark bulunabilir 7. </li><li> Uygun anestezi sonrasında, sıcaklık kontrollü cerrahi masaya hayvan koyun (yukarı uzunluğu 3 inç) karın orta hat kesi yapmak ve vena kava ve aorta ortaya çıkarmak. </li><li> Çölyak ve superior mezenterik arterler ve abdominal etrafında bitişik harfleri yerleştirinyukarıda aort; Arter yok. </li><li> Renal arterlerin altında abdominal aorta incelemek ve çevresinde iki bitişik harfler koyun, ancak lige yok künt, daha sonra bitişik harfler üzerinde aorta kelepçe ve alt iplik kravat. </li><li> Kelepçe altında (PBS ile dolu bir şırınga pompası takılı) bir polietilen PE50 boru ile aort kateter ve ikinci dikilen ile kateter düzeltmek; , kelepçe kaldırmak pompa açın ve aorta bağlanır ve çölyak arterler ile mezenterik. Hızla baskıyı hafifletmek için renal ven kesi yapmak. </li><li> / Dakika 6 ml &#39;lik bir oranda, 2 veya 3 dakika için önceden soğutulmuş PBS ile aort infüze. </li><li> Perfüzyon, rüsumat durdurun ve 7 böbrekler açarlar ve PBS çözüm buz üzerine koydu. IACUC tarafından onaylanmış protokole göre hayvan Euthanize. </li></ol> Sıçan glomerulusların 2. izolasyonu <ol><li> RPMI 1640 içinde% 5 BSA, taze çözelti 30 ml hazırlayın. </li><li> Bir jilet ve makas kullanarak,Her iki böbreğin korteks izole ve ardından homojen hale gelinceye kadar kıyma. Bu prosedür, önceki 7 tarif edilmiştir. </li><li> 100 gözlü paslanmaz çelik elek içinden geçen aşaması esnasında kıyılmış doku itin bir spatula kullanılarak (BSA / RPMI çözeltisi% 5 önceden ıslatılmış). Akış ve yer çekimi kuvveti ile akış ile 140 ağ elek boyunca geçmesine izin toplayın. </li><li> Bir ön-ıslatılmış 200 gözenekli elek kullanılarak bir elek 140 ağ toplanan akış geçirgen filtre süzüntü atmak ve 10, elek üst yıkama - ilgili tortu glomerül toplamak için hazırlanmış BSA / RPMI çözeltisi 15 ml Elek. </li><li> Kadar 20 dakika boyunca tüpün altındaki glomerüller tortu 15 ml tüp buz üzerinde glomerül BSA / RPMI çözeltisi koyun ve bildirin. Tüpün alt glomerüller konsantre açıkça görülecektir. Tüp içinde yaklaşık olarak 2 ml bırakarak, fazla çözelti çıkarın. </li></ol> 3. Tek kanallı Patch-kelepçe Electrophysiology <ol><li> MW 70.000 ile kaplanarak 5 x 5 mm kapak cam cips hazırlayın - 150.000 poli-ʟ-lizin ve kurumaya bırakın. Kapak cam başına su içinde% 0.01 steril filtre edilmiş çözeltisi, yaklaşık 30 ul kullanın. </li><li> RT deneysel çözümler Isınma ve patch-kelepçe odasını ve pipet doldurun. TrpC kanalları izleme, mM banyo solüsyonu kullanın: 126 NaCl, 1 CaCl2, 10 HEPES, 2 MgCl2, 10 glukoz, pH 7.4; Pipet: 126 NaCl, 1.5 CaCl2, 10 HEPES, 10 glukoz; pH 7.4. <ol><li> Tavsiye çalışmalarda (ilgili olmayan endojen kanal aktivitesini engellemek için pipet solüsyonuna inhibitörleri ekleme vardır: 100 uM niflumik asit ya da DIDS (Ca2 + ile aktive edilen Cı engellemek için - kanalları), 10 mM TEA (inhibe yüksek ölçüde iletkenliğe sahip Ca2 + - bağımlı K + kanalı), 10 nM iberiyotoksin (,) 10 uM nikardipin Ca2 + ile aktive edilen K + kanallarını bloke etmek (N-tipi Ca engellemek2+ doğrudan patch-kelepçe deneyden önce kanalları)). </li></ol></li><li> Yavaşça glomerüller içeren çözüm karıştırmak ve sonra poli-ʟ-lizin kaplı kapak cam cips bunun yaklaşık 50 ul uygulayın. Glomerül yaklaşık 5 dakika süreyle tutunma olsun. </li><li> Patch-kelepçe odasına banyo solüsyonu ile önceden doldurulmuş için glomerül cam cips taşı; serbest glomerül kaldırılmasını sağlamak için 1 dakika için / dakika 3 ml bir oranda bölme serpmek. </li><li> Bir hücre ekli modunda 7 geleneksel bir yama-kelepçe deney. Bir cam pipet ile, (7-10 M? Pipet direnci), bir pipet ve hafif bir emme (izole glomerulus yüzeyi üzerinde bir podosit bağlanmış bir pipet uygulayarak podosit membran arasında yüksek dirençli bir yalıtım oluşturmak ilgili, Şekil 2&#39;de gösterildiği gibi olduğu sol). </li><li> Hücre bağlı ölçümler için, sekiz kutuplu Bessel filtresi ile 300 Hz akımları düşük geçmektedir. </li><li> U6 saat - se kadar 4 için patch-kelepçe deneylerinde glomerüller izole. Buz üzerinde stok glomerüller fraksiyonu tutun. </li></ol> Podositler hücre içi kalsiyum konsantrasyonu 4. oranlı metrik Konfokal floresans ölçümleri <ol><li> Konik tüp, 0.5 ml (2.5 açıklanan) glomerüller fraksiyonu 500 ul yerleştirin ve kalsiyum boyalar Fura Kırmızı, AM ve Fluo-4, AM ekleyin. 2 mm ve (DMSO içinde çözülmüş -20 ° C&#39;de saklayın) Fura Kırmızı, AM ve Flu-4, GM, sırasıyla 1 mM stok konsantrasyonları kullanımı ve glomerüller fraksiyonunun 500 ul her bir boya 2.5 ul kullanın. Hemen boyaların ilave edilmesinden sonra, alüminyum folyo ile tüp kapsamaktadır. </li><li> Oda sıcaklığında 1 saat için en az 20 dakika kadar bir döner çalkalayıcı üzerinde yer borular. Not: Farmakolojik maddeler, bu aşama sırasında eklenebilir. </li><li> Cam lamelleri hazırlanması poli-ʟ-lisin ile kapak ve 70 ° C&#39;ye kadar ısıtıldı levha seti kullanarak kurutma sağlar. </li><li> Kalsiyum boyalar yükleme komplet sonraE, poli-ʟ-lizin kaplı kapak çözeltiyi içeren glomerül 100 ul uygulamak ve bununla ilgili olarak, 5 dakika için yüzeye dönelim. Bir görüntüleme odasına glomerüller bağlı lamelleri monte edin ve banyo çözeltisi (() mM olarak ihtiva eden: MgCl2 145 NaCl, 4.5 KCl, 2, 10 HEPES, pH 7.35) ile serpmek 3 ml / dk hızda kaldırmak için bekâr glomerüller ve kalan boyalar. </li><li> Bir 488 nm eksitasyon dalga boyu ve radyasyon filtreleri (sırasıyla Fluo-4 ve Fura Red için 525/25 ve 650/25 nm) konfokal lazer tarama mikroskobu ayarlayın. İstediğiniz bir frekans ve çözünürlük görüntüleme yazılımı ayarlayın. </li><li> Aydınlık içinde glomerüller bulun ve sonra floresan sinyalinin tespiti açın. Sinyalin doygunluk önlemek için her boya için lazer yoğunluğunu ayarlar. Doğrudan cama bağlı Podositler ile odak düzlemi seçin. Bu ilaca yanıt olarak bir glomerulus büzülmesiyle kaynaklanan etkiyi en aza indirirUygulama. Seçim glomerulus iyi cama takılı olduğundan çift çek; </li><li> Seçim odak düzlemi görüntüleme başlayın, (yüksek çözünürlüklü görüntü için 60X / NA 1.4 veya benzer objektif lens kullanın. Hızlı Ca 2+ geçici değişiklikleri görmenizi 4 sn set frekans ile 512 x 512 görüntü almak) ilgi ilaçları uygulamak ve yanıtı kaydedin. <ol><li> (Glomerül yüzeyinde Podositler görüntülenmesini sağlamak için, mümkün olduğu kadar camın yüzeyine yakın olarak) arzu edilen bir odak düzlemi seçin. Fluo4 ve FuraRed kanallarda floresan yoğunluğunu kontrol edin ve glomerulus açıkça aydınlık görülür olduğundan emin olun. </li><li> Görüntüleme başlatın. Herhangi bir ilaç uygulamasından önce, bazal floresans rekor en az 1 dakika sinyali (hiçbir ani sivri veya sinyal solma) kararlı olduğundan emin olmak için. </li><li> Bir mikropipet yardımıyla istenilen ilaçları uygulayın; dikkatli olmak ve ilaç iyi diffüz başardı sağlamak ve glomer ulaşmakulus. Gerekirse, seçilen odak düzlemi izlemek ve çünkü ilaç uygulamasının odak dışına taşımak olmadığını kontrol ediniz hafifçe banyo çözüm karıştırın. </li><li> Fluo4 ve FuraRed sinyalleri için flüoresan yoğunluğundaki değişimlerle kaydedin. Kayıt sinyal plato seviyesine ulaştığında veya bir zirveye ulaşır ve daha sonra bazal dönene kadar bekleyerek, yeterince uzun olduğundan emin olun. Gerekirse Çözelti değişiklik ya da başka herhangi bir ilaç ilave gerçekleştirin. </li><li> Kaydı durdurmak ve yazılım özgün biçiminde dosyayı kaydedin. </li></ol></li></ol> Kalsiyum Ölçümleri 5. Görüntü Analizi <ol><li> Yerli ND2 formatındaki görüntüleri içe izin veren ND Programı eklentisi ile donatılmış ImageJ açık yazılım ile görüntü analizi yapın. </li><li> Görüntü dizisi İthalat; Kanal bölmek ve bir hyperstack gri tonlama modu kullandığınızdan emin olun. </li><li> O kadar ImageJ yazılımı analiz → Araçlar → ROI yöneticisi yolunu izleyinkalem ROI yöneticisi penceresi. Oval seçim aracını ve ROI Yöneticisi &quot;Add (t)&quot; fonksiyonunu kullanarak faiz (Podositler) birkaç bölgeleri seçin. Son ROI olarak arka planda alanı seçin; (→ Kaydet fazla) seçilen ROI kaydedin. </li><li> Analizler için seçilen kanalı içeren pencereyi vurgulayın. , YG Yöneticisi Çoklu Ölçüm işlevi → Daha kullanın dışarı çıkar diyalog içinde &quot;tek satır dilim başına&quot; kutusunu işaretleyin ve sonra Tamam &#39;ı tıklatın. seçeneğini ayarlayın Ölçümleri → menü seçeneği Sonuçlar girmek &quot;gri Ortalama değer&quot; ve görüntülenir her ROI için piksel yoğunluğu değerlerini hesaplamak: Sonuçlar Sonuçları penceresinde ayarlanabilir formatında gösterilir Her ROI için ayrı sütunlarda Sonuçları penceresi. </li><li> Tercih veri analiz yazılımı içine her bir kanal (Fluo-4 ve Fura Kırmızı) için ölçülen ROI yoğunluk değerlerini kopyalayın; Her bir DAT dan çıkarma arka plan yoğunluğu değerleribir nokta. </li><li> Her bir zaman noktası için, Fura kırmızı kanallara Flu-4 yoğunluklarının oranı hesaplanır. Plot dağılım / her ROI için Ca 2+ geçici hat noktası zamanı değişir. Seçilen glomeruli için ortalama / SE değerlerini hesaplamak. Not: Zaman sütunu 4,5 seçilen görüntüleme frekansına göre ayarlanması gerekir. </li></ol> Flu-4 Floresan Sinyal kullanma 6. Hücre içi kalsiyum Konsantrasyon Hesaplamaları <ol><li> Glomerulusların bir örneğini alın ve 4.7 adıma deneysel protokol yukarı gerçekleştirin. Eşiğe kaydından sonra banyo çözeltisine ve kayıt floresan yoğunluğu artışı (10 uM olmalıdır banyo odasında nihai konsantrasyon) İyonomisin ekleyin. Yoğunluk maksimuma ulaşır ve çürüme başladığında, MnCI2 eklemek floresan 8 gidermek için (nihai konsantrasyon 5 mM olmalıdır). </li><li> 6.1 elde edilen verileri analiz edin. Elde edilen Flu-4 sinyal ROI yoğunluğu değerleri doğrultusunda kopyalamaTercih analiz yazılımı ile 5,1-5,5 açıklanan protokole. </li><li> Bir formül kullanılarak ROI karşılık gelen Podosit olarak (nM) hücre içi kalsiyum konsantrasyonunu hesaplayın: <img alt="Denklem 1" src="/files/ftp_upload/52850/52850equation1.jpg" /> Kd Fluo-4 (345 nM) sabit, önceden belirlenmiş bir ayrılma olduğu, F (taban) için kalsiyum konsantrasyonunun hesaplanması olan zaman noktasında yoğunluğudur ve F dakika ve Fmax de yoğunluk değerleri (iyonomisin uygulama sonrası) ve (MnCI2) ile flöresan soğutmadan sonra, maksimum kalsiyum yük noktası, sırasıyla, (Şekil 3&#39;e bakınız). </li></ol>

<ol>
	<li>Machuca, E., Benoit, G., Antignac, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+nephrotic+syndrome:+connecting+molecular+genetics+to+podocyte+physiology.">Genetics of nephrotic syndrome: connecting molecular genetics to podocyte physiology.</a> Hum. Mol. Genet. 18, R185-R194 (2009).</li><li>Haraldsson, B., Nystrom, J., Deen, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+glomerular+barrier+and+mechanisms+of+proteinuria.">Properties of the glomerular barrier and mechanisms of proteinuria.</a> Physiol. Rev. 88, 451-487 (2008).</li><li>Patrakka, J., Tryggvason, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+role+of+podocytes+in+proteinuria.">New insights into the role of podocytes in proteinuria.</a> Nat. Rev. Nephrol. 5, 463-468 (2009).</li><li>Savin, V. J., Sharma, R., Lovell, H. B., Welling, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+albumin+reflection+coefficient+with+isolated+rat+glomeruli.">Measurement of albumin reflection coefficient with isolated rat glomeruli.</a> J. Am. Soc. Nephrol. 3, 1260-1269 (1992).</li><li>Gloy, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+depolarizes+podocytes+in+the+intact+glomerulus+of+the+Rat.">Angiotensin II depolarizes podocytes in the intact glomerulus of the Rat.</a> J. Clin. Invest. 99, 2772-2781 (1997).</li><li>Nitschke, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+increases+the+intracellular+calcium+activity+in+podocytes+of+the+intact+glomerulus.">Angiotensin II increases the intracellular calcium activity in podocytes of the intact glomerulus.</a> Kidney Int. 57, 41-49 (2000).</li><li>Ilatovskaya, D., Staruschenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-channel+analysis+of+TRPC+channels+in+the+podocytes+of+freshly+isolated+glomeruli.">Single-channel analysis of TRPC channels in the podocytes of freshly isolated glomeruli.</a> Methods Mol Biol. 998, 355-369 (2013).</li><li>Snitsarev, V. A., McNulty, T. J., Taylor, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+heavy+metal+ions+perturb+fura-2+measurements+of+basal+and+hormone-evoked+Ca2++signals.">Endogenous heavy metal ions perturb fura-2 measurements of basal and hormone-evoked Ca2+ signals.</a> Biophys. J. 71, 1048-1056 (1996).</li><li>Fukuda, A., Fujimoto, S., Iwatsubo, S., Kawachi, H., Kitamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mineralocorticoid+and+angiotensin+II+receptor+blockers+on+proteinuria+and+glomerular+podocyte+protein+expression+in+a+model+of+minimal+change+nephrotic+syndrome.">Effects of mineralocorticoid and angiotensin II receptor blockers on proteinuria and glomerular podocyte protein expression in a model of minimal change nephrotic syndrome.</a> Nephrology (Carlton). 15, 321-326 (2010).</li><li>Abramowitz, J., Birnbaumer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+and+pathophysiology+of+canonical+transient+receptor+potential+channels).">Physiology and pathophysiology of canonical transient receptor potential channels).</a> FASEB J. 23, 297-328 (2009).</li><li>Heeringa, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+TRPC6+mutation+that+causes+childhood+FSGS.">A novel TRPC6 mutation that causes childhood FSGS.</a> PLoS ONE. 4, e7771 (2009).</li><li>Zhang, X., Song, Z., Guo, Y., Zhou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+role+of+TRPC6+in+vitamin+D+ameliorating+podocyte+injury+in+STZ-induced+diabetic+rats.">The novel role of TRPC6 in vitamin D ameliorating podocyte injury in STZ-induced diabetic rats.</a> Mol. Cell. Biochem. 399, 155-165 (2015).</li><li>Bohrer, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+the+puromycin-induced+defects+in+the+transglomerular+passage+of+water+and+macromolecules.">Mechanisms of the puromycin-induced defects in the transglomerular passage of water and macromolecules.</a> J. Clin. Invest. 60, 152-161 (1977).</li><li>Olson, J. L., Rennke, H. G., Venkatachalam, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+the+charge+and+size+selectivity+barrier+of+the+glomerular+filter+in+aminonucleoside+nephrosis+in+rats.">Alterations in the charge and size selectivity barrier of the glomerular filter in aminonucleoside nephrosis in rats.</a> Lab. Invest. 44, 271-279 (1981).</li><li>Schiessl, I. M., Castrop, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+AT2+receptor+activation+attenuates+AT1+receptor-induced+increases+in+the+glomerular+filtration+of+albumin:+a+multiphoton+microscopy+study.">Angiotensin II AT2 receptor activation attenuates AT1 receptor-induced increases in the glomerular filtration of albumin: a multiphoton microscopy study.</a> Am J Physiol Renal Physiol. 305, F1189-F1200 (2013).</li><li>Ilatovskaya, D. V., Levchenko, V., Ryan, R. P., Cowley, A. W., Staruschenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NSAIDs+acutely+inhibit+TRPC+channels+in+freshly+isolated+rat+glomeruli.">NSAIDs acutely inhibit TRPC channels in freshly isolated rat glomeruli.</a> Biochem. Biophys. Res. Commun. 408, 242-247 (2011).</li><li>Peti-Peterdi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+wave+of+tubuloglomerular+feedback.">Calcium wave of tubuloglomerular feedback.</a> Am. J. Physiol. Renal Physiol. 291, F473-F480 (2006).</li><li>Peti-Peterdi, J., Warnock, D. G., Bell, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+directly+stimulates+ENaC+activity+in+the+cortical+collecting+duct+via+AT(1)+receptors.">Angiotensin II directly stimulates ENaC activity in the cortical collecting duct via AT(1) receptors.</a> J. Am. Soc. Nephrol. 13, 1131-1135 (2002).</li><li>Ilatovskaya, D. V., Palygin, O., Levchenko, V., Staruschenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+characterization+of+the+P2+receptors+profile+in+the+podocytes+of+the+freshly+isolated+rat+glomeruli.">Pharmacological characterization of the P2 receptors profile in the podocytes of the freshly isolated rat glomeruli.</a> Am. J. Physiol. Cell Physiol. 305, C1050-C1059 (2013).</li><li>Ilatovskaya, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+has+acute+effects+on+TRPC6+channels+in+podocytes+of+freshly+isolated+glomeruli.">Angiotensin II has acute effects on TRPC6 channels in podocytes of freshly isolated glomeruli.</a> Kidney Int. 305, C1050-C1059 (2014).</li><li>Schaldecker, T., et al. <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+the+TRPC5+ion+channel+protects+the+kidney+filter.">Inhibition of the TRPC5 ion channel protects the kidney filter.</a> J. Clin. Invest. 123, 5298-5309 (2013).</li><li>Roshanravan, H., Dryer, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+acting+through+P2Y+receptors+causes+activation+of+podocyte+TRPC6+channels:+role+of+podocin+and+reactive+oxygen+species.">ATP acting through P2Y receptors causes activation of podocyte TRPC6 channels: role of podocin and reactive oxygen species.</a> Am. J. Physiol. Renal Physiol. 306, F1088-F1097 (2014).</li><li>Anderson, M., Roshanravan, H., Khine, J., Dryer, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+activation+of+TRPC6+channels+in+rat+podocytes+requires+generation+of+reactive+oxygen+species.">Angiotensin II activation of TRPC6 channels in rat podocytes requires generation of reactive oxygen species.</a> J. Cell. Physiol. 229, 434-442 (2014).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Burada tarif edilen bir yaklaşım, kemirgen glomerül Podositler kalsiyum işleme analizi sağlar. Bu teknik, patch-kelepçe tek kanallı elektrofizyoloji ve floresan rasyometrik konfokal görüntüleme uygulama sağlar. Bununla birlikte, her iki yaklaşım da kendi ayrı ayrı kullanılabilir. Önerilen protokol 1) böbrek temizleme dahil olmak üzere birçok nispeten basit adımları vardır; 2) diferansiyel eleme ile glomerül izolasyonu; 3) patch-kelepçe elektrofizyolojik deneyler veya hücre içi kalsiyum değiş...

Böbrekler, çeşitli maddeler için homeostatik dengeyi korumak ve toplam kan basıncını belirleyen bir şekilde kan hacmini düzenler. Hiper veya hipotansiyon arasında değişen renal filtrasyon, geri emiliminin veya salgı kurşun bozukluklar veya eşlik patolojik durumlar, sonunda böbrek nakli gerektiren son dönem böbrek hastalığı için. kapiller endotel, bazal membran ve epitel hücreleri tek-hücre tabakası - - yarık diyafram bütünlüğü ve işlevi 1 korunmasında önemli bir rol oynamaktadır Podositler renal filtreleme birimi (glomerulus) üç tabakadan oluşur. Permselektif glomerüler filtre Disfonksiyon gibi proteinüri olarak makromoleküllerin, idrar kaybına neden olur. Çeşitli ajanlar glomerüller filtrasyon bariyerinin bütünlüğünü belirleyen Podositler ve ayak süreçleri, yapısını etkileyebilir. Podositler glom bakım katılaneruli filtrasyon işlevi. Bu Podosit tarafından uygunsuz kalsiyum taşıma hücre hasarı yol açar ve nefropatilerin 2,3 çeşitli biçimlerde ilerlemesinde önemli bir rol oynadığı tespit edilmiştir. Bu nedenle, podosit fonksiyonunun çalışmalar için etkili olacaktır hücre içi kalsiyum konsantrasyonu değişiklikleri, doğrudan ölçümü için izin veren bir modelinin geliştirilmesi. İzole glomerüller önce albümin yansıma katsayısı ölçümü 4 ve bütün hücre elektrofizyolojik patch-kelepçe ölçümlerde 5,6 integral hücresel akımların değerlendirmesini değiştirir dahil çok sayıda çalışmalarda kullanılmıştır. Mevcut yazıda, farmakolojik ajanların uygulamalarına tepki olarak hücre içi kalsiyum konsantrasyonu değişiklikleri ölçmek hücrelerin içinde kalsiyum bazal düzeylerini tahmin ve bireysel kalsiyum kanalları aktivitesini değerlendirmek için araştırmacı izin veren protokol açıklar. Ratometric kalsiyum konsantrasyonu ölçümleri ve patch-kelepçe electrophysiology sırasıyla podosit ve kanal aktivitesi olan hücre içi kalsiyum konsantrasyonu değişiklikleri belirlemek için kullanıldı.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Fluo4 AM</td>
			<td>Life Technologies</td>
			<td>F14217</td>
			<td>500&#181;l in DMSO</td>
		</tr>
		<tr>
			<td>FuraRed AM</td>
			<td>Life Technologies</td>
			<td>F-3020</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic acid</td>
			<td>Sigma-Aldrich</td>
			<td>F-68&#160;</td>
			<td>solution</td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>I3909-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube rotator</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-090-753</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon confocal microscope (inverted)</td>
			<td>Nikon</td>
			<td>Nikon A1R</td>
			<td>&#160;Laser exitation 488nm. Emission filters 500-550nm and 570-620nm</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Nikon</td>
			<td>Plan Apo 60x/NA 1.4 Oil</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass</td>
			<td>Thermo Scientific</td>
			<td>6661B52</td>
			<td></td>
		</tr>
		<tr>
			<td>High vacuum grease</td>
			<td>Dow Corning</td>
			<td></td>
			<td>Silicone Compound</td>
		</tr>
		<tr>
			<td>Software</td>
			<td>Nikon</td>
			<td>Nikon NIS-Elements&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording/perfusion chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Patch Clamp amplifier</td>
			<td>Molecular Devices</td>
			<td>MultiClamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition System</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td>Axon Digidata&#174; System</td>
		</tr>
		<tr>
			<td>Low Pass Filter</td>
			<td>Warner Instruments</td>
			<td>LPF-8</td>
			<td>8 pole Bessel</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B150F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument Co</td>
			<td>P-97</td>
			<td>Flaming/Brown type micropipette puller</td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>MF-830</td>
			<td>Japan</td>
		</tr>
		<tr>
			<td>Motorized Micromanipulator</td>
			<td>Sutter Instrument Co</td>
			<td>MP-225</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvibration isolation table</td>
			<td>TMC</td>
			<td></td>
			<td>equipped with Faraday cage</td>
		</tr>
		<tr>
			<td>Multichannel valve perfusion system</td>
			<td>AutoMake Scientific</td>
			<td>Valve Bank II</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording/perfusion chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>Molecular Devices</td>
			<td>pClamp 10 . 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicardipine</td>
			<td>Sigma-Aldrich</td>
			<td>N7510</td>
			<td></td>
		</tr>
		<tr>
			<td>Iberiotoxin</td>
			<td>Sigma</td>
			<td>I5904-5UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Niflumic acid</td>
			<td>Sigma-Aldrich</td>
			<td>N0630</td>
			<td></td>
		</tr>
		<tr>
			<td>DIDS</td>
			<td>Sigma-Aldrich</td>
			<td>D3514-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>TEA chloride</td>
			<td>Tocris</td>
			<td>T2265</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11835030</td>
			<td>without antibiotics</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A8327</td>
			<td>30% albumin solution</td>
		</tr>
		<tr>
			<td>Temperature controlled surgical table&#160;</td>
			<td>MCW core</td>
			<td></td>
			<td>for rodents</td>
		</tr>
		<tr>
			<td>Steel sieves:</td>
			<td></td>
			<td></td>
			<td>#100 (150 &#956;m), 140 (106 &#956;m)</td>
		</tr>
		<tr>
			<td>Gilson, Inc&#160; SIEVE 3 SS FH NO200</td>
			<td>Fisher Sci</td>
			<td>50-871-316</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson, Inc&#160; SIEVE 3 SS FH NO270</td>
			<td>Fisher Sci</td>
			<td>50-871-318</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson, Inc&#160; SIEVE 3 SS FH NO400</td>
			<td>Fisher Sci</td>
			<td>50-871-320</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;mesh 200&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>s4145</td>
			<td>screen for CD-1</td>
		</tr>
		<tr>
			<td>Binocular microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular microscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump-based perfusion system</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polyethylene tubing</td>
			<td>Sigma-Aldrich</td>
			<td>PE50</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane anesthesia</td>
			<td><a href="http://www.vetequip.com/">http://www.vetequip.com/</a></td>
			<td>911103</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Other basic reagents</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-channel analysis and calcium imaging in the podocytes of the freshly isolated glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Burada Podositler kalsiyum seviyeleri akut değişiklikler ölçülerek değerlendirilmesi amaçlanmıştır. 1, taze bir Podositler yüksek çözünürlüklü canlı floresan konfokal görüntüleme ve tek iyon kanalı aktivitesi kayıtları gerçekleştirmek için tasarlanan deney protokolünün şematik bir temsilini göstermektedir, Şekil İzole kemirgen glomerüller. Sıçan anestezi sonra Kısaca, böbrekler kan bunları temizlemek için PBS ile yıkanmalıdır. Ardından, böbre...

Watch this Scientific Journal Video about Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli at JoVE.com

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

Taze İzole glomerulusların Podositler Tek kanallı Analizi ve Kalsiyum Görüntüleme

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

single-channel-analysis-calcium-imaging-podocytes-freshly-isolated

Research

JoVE Journal

Biology

10.9K Görüntüleme.  Medical College of Wisconsin. Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli. 

Video: Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

FDG micro-PET/CT ile Fare Brown Fat Fonksiyonel Görüntüleme

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Biyoloji

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Apoptotik hücresi Temizliği görüntülenmesi sırasında Canlı<em&gt; Drosophila</em&gt; Embriyo

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Kalsiyum değerlendirilmesi Bozulmamış İskelet kas liflerinin içinde Sparks

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

İzole Kan-perfüze Sıçan Akciğer Hazırlık Tek Mikrodamar Geçirgenliği miktarının

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Ozmotik Su Geçirgenliği Katsayısı (P Ölçme<sub&gt; F</sub&gt;) Küresel Hücreleri: Bir Örnek olarak İzole Bitki protoplastlan

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Patch Kelepçe ve Canlı Floresan Mikroskobu uygulanması Taze İzole PKD Epitelinin Fonksiyonel Properties Monitör

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Yüksek çözünürlüklü Time-lapse Görüntüleme ve Yaşayan İnsan Umbilikal Ven Endotelyal Hücrelerinde Mikrotubul Dinamiği Otomatik Analizi

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

Fare İzole perfüze Böbrek Tekniği

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Zebra balığı adacıkları tek hücreli çözünürlük kalsiyum Imaging'i kullanma Beta hücre fonksiyonu Analizi

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Hücre içi kalsiyum ve taze izole ve olduğu gibi fare serebral endotel membran potansiyeli eşzamanlı ölçümler

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...