Biz tesisleri ve iyon kanalları çalışmaları için mükemmel bir ortam sağlamak için Fizyoloji Anabilim Dalı ve Membran Biyoloji, University of California Davis müteşekkiriz.

1. Dinlenme İnsan T Lenfositler hazırlanması <ol><li> RosetteSep İnsan T Hücre Zenginleştirme Kokteyl ve RosetteSep Yoğunluk Orta kullanarak, üreticinin talimatlarına göre insan kanı örnekleri T hücreleri arındırmak. Ortaya çıkan hücre nüfusun% 95 CD3 + dinlenme T hücreleri içermelidir. Biz insan T lenfositleri sağlıklı gönüllülerin UC Davis İç Değerlendirme Kurulu tarafından onaylanan protokol uyarınca alınan periferik kan örnekleri arındırmak. </li><li> Izolasyondan sonra,% 10 fetal buzağı serumu,% 2 GlutaMAX-I,% 1 RPMI 1640 ile desteklenmiş, glutamin ve Hepes RPMI-1640 ortamı içeren hücre kültür ortamında 0.5 x 10 6 hücre / mL yoğunluğu dinlenme T hücreleri tekrar süspansiyon vitamin çözeltisi,% 1 RPMI 1640 amino asitler çözüm,% 1 sodyum piruvat, ve% 0.03 β-mercaptoethanol. </li><li> Place hücre hücre kültürü şişeler içine süspansiyon ve 37 ° C nemlendirilmiş bir inkübatör% 5 CO 2 ile 24 saat kadar önce deneme için korumak. </li></ol> 2. Kayıt Odası hazırlanması <ol><li> Aşağıdaki kayıt odaları hazırlaması gerekli:) b, a) silikon O-halkalar yuvarlak,% 70 etanol ve distile H 2 O, c ile temizlenmelidir 25 mm lamelleri), Sylgard184 karışık üreticinin talimatlarına göre hazırlanan, ve d) iki adet 60 x 15 mm Petri kapları. </li><li> Yeri ~ 60x15 mm Petri kabında 0,5 mL içine karışık Sylgard184. </li><li> O-ring alt kısmında Sylgard184 kullanarak forseps içine batırın. </li><li> O-ring koyarak başka bir Petri kabı temiz bir yüzey, O-ring kenarına aşırı Sylgard184 çıkarın. </li><li> Halka lamel üzerine yerleştirin ve aşağı doğru bastırın. </li><li> 85 ° C 40-60 dk ya Sylgard184 kadar polimerize odaları ısıtarak Sylgard184 Cure. </li><li> Kayıt odaları tozsuz bir kapta saklayın. </li></ol> 3. Patch Pipetler hazırlanması <ol><li> Deney günü, cam kapiller ve bir pipet çektirmenin kullanarak bir ~ 2-mikron ucu çapı pipetler çekin. Biz filament (1.5 mm ve ID: 1.10 mm OD) ile borosilikat tüp kullanın. Tozsuz bir yerde saklayın pipetler. </li><li> Standart bir prosedür 9 göre Sylgard184 veya HIPEC R6101 Semiconductor Koruyucu Kaplama Coat pipet ipuçları. </li><li> Deneyden önce hafifçe pipetler yangın lehçe. </li></ol> 4. Patch Kelepçe Kurulum Hazırlığı <ol><li> Patch-klemp amplifikatör kontrol yazılımı gigaseal oluşumu için makroları yapılandırma ve kaydedin. Biz Windows XP ve veri toplama için nabız yazılım tarafından desteklenen bir EPC-10 amplifikatör kullanır. EPC-10 kılavuzuna göre, &quot;hücre&quot; ve &quot;tam hücreli&quot; makrolar &quot;set-up&quot; ayarlayın ve tüm deneyler için bunları kullanmak. </li><li> Deneme öncesinde banyo çözümleri ve Tablo 1&#39;de yer alan pipet çözümleri hazırlayın ve 1 hafta kadar 4 ° C sıcaklıkta saklayın. </li></ol><table border="1"><tbody><tr><td> Kimyasallar </td><td colspan="3"> Banyo Çözümleri (mM) </td><td> Pipet Çözümleri (mM) </td></tr><tr><td></td><td> # 1 </td><td> # 2 </td><td> # 3 </td><td></td></tr></tbody><tbody><tr><td> CH 3 SO 3 Na </td><td> 130 </td><td> 110 </td><td> 125 </td><td> - </td></tr><tr><td> NaCl </td><td> 2 </td><td> 4 </td><td> 5 </td><td> - </td></tr><tr><td> Ca (OH) 2 </td><td> - </td><td> 20 </td><td> - </td><td> - </td></tr><tr><td> MgCl 2 </td><td> 3 </td><td> 1 </td><td> - </td><td> 5 </td></tr><tr><td> MgSO 4 </td><td> - </td><td> - </td><td> - </td><td> 2 </td></tr><tr><td> Hepes </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 15 </td></tr><tr><td> HEDTA </td><td> - </td><td></td><td> 10 </td><td> - </td></tr><tr><td> EDTA </td><td> - </td><td> - </td><td> 1 </td><td> - </td></tr><tr><td> Cs-spartate </td><td> - </td><td> - </td><td> - </td><td> 125 </td></tr><tr><td> BAPTA </td><td> - </td><td> - </td><td> - </td><td> 12 </td></tr><tr><td> Glikoz </td><td> 10 </td><td> 10 </td><td> 10 </td><td> - </td></tr></tbody></table> Tablo 1. Tam hücreli Carc mevcut kayıtları için çözümler. Bütün kimyasallar Sigma-Aldrich, St Louis, MO satın alınmıştır. <ol start="3"><li> Yama pipet çözüm ve her banyo çözümü 10 arasında sıvı birleşme potansiyelleri (LJ) belirleyin. LJ değeri satın alma yazılımı kullanarak uygun bir yere kaydedin. Örneğin, Darbe programı &quot;Amplifikatör&quot; penceresinde &quot;LJ&quot; kontrol LJ değerini yerleştirin. </li><li> Şekil 1&#39;de gösterildiği gibi deneyler önce satın alma yazılımı uygun yerde stimülasyon protokolleri, yapılandırma ve kaydedin. Darbe programında, &quot;Darbe Üretimi&quot; penceresinde satın alma protokolü ayarlayın. <img src="/files/ftp_upload/2346/2364fig1.jpg" alt="Şekil 1" /> Şekil 1. Uyarım protokolü -120 süresi 50 ms +100 mV Gerilim rampası her 0.2 - 2 (5 - 0,5 Hz) uygulanır. Rampaları arasındaki Holding potansiyel (V s) +30 mV korunur. </li></ol> 5. Mikroskop Sahne Montaj Hücreler <ol><li> Hücreleri kaplama önce sonra ~ 20 dakika ve yıkama ve H 2 O ile Hank dengeli tuz solüsyonu (HBSS), poli-L-lizin ile Coat kayıt odaları . </li><li> 37 odası ve 20 dakika boyunca inkübe 0.2-0.5 x10 6 hücre içeren hücre süspansiyonu Plaka 500 mcL ° C </li><li> Bir oda sahibinin bağlı hücreleri ile kayıt odasında sabitleyin. Biz özel yapılmış bir odasının taban lamel alt destekler tutucu, lamel üst karşı üst kısmı hafifçe presler. </li><li> Inverted mikroskop sahnede oda sahibinin yerleştirin. </li><li> Iletilen ışık hücreleri üzerinde odaklanın. Biz 40X immersiyon yağı hedefi kullanın. </li><li> Ag / AgCl referans elektrot banyo içine yerleştirin. </li><li> Multibarrel, yerçekimine bağlı perfüzyon sistemi hizalayın. Girişinin tüpünün ucu, 45 derecelik bir açıyla görüş alanı lamel yakın yerleştirilmelidir. </li><li> Girişi tüp ucu karşısında emme borusu yerleştirin. </li><li> Odasına serpmek fosfat tamponlu salin (PBS) veya HBSS hücre kültürü medya ve gevşek bağlı hücreleri yıkayacak. </li></ol> 6. Kerek Kanal Kayıt Akımlar <ol><li> Lamel sıkıca bağlı bir hücre. Biz genellikle pürüzsüz bir dış yüzeye sahip ortalama büyüklükteki bir yuvarlak hücre seçin. </li><li> Ca 2 +-free, 3 mM Mg 2 + içeren banyo çözüm SERCA pompa engellemek için 8-10 dakika süreyle 0.5-1 mcM thapsigargin içeren # 1 (Tablo 1) ile kayıt odasında serpmek. Biz mağaza gigaseal oluşumu öncesinde tüketmek için bu adımı gerçekleştirmeniz. </li><li> Eppendorf Microloader ve P-20 pipeti kullanarak, yama pipet pipet çözüm (Tablo 1) ile doldurun ve amplifikatör headstage pipet tutucu içine yama pipet yerleştirin. Ca 2 + şelasyon BAPTA pasif mağaza tüketmek ve Ca 2 +-bağımlı Kerek kanal inaktivasyon azaltmak için pipet çözümü yer almaktadır. </li><li> Banyo yama pipet içine girmeden önce küçük bir miktar pozitif basınç (H 2 O ~ 3 inç) uygulayın. Biz bir yama pipet içinde pozitif veya negatif basınç oluşturmak için basınçlı hava ve vakum hatları bağlı bir ölçüye göre basınç emme aygıtı kullanın. </li><li> Mikroskop üzerinden görsel kontrol altında banyoya yama pipet indirin. &quot;Osiloskop&quot; penceresinde, geçerli bir pipet akan göreceksiniz. Geçerli genliği bir adım gibi değişiklik, önceden ayarlanmış makro tarafından uygulanan küçük bir genlik gerilim darbesi (test pulse) tarafından indüklenen olmalıdır. Şu anda, pipet direnç değeri belirlemek mümkün olmalıdır. Bizim pipetler direnç genellikle M 5-6. </li><li> Pipet ve referans elektrot arasında oluşturulan &quot;offset&quot; potansiyel düzeltin. </li><li> Hücre zarının dokunana kadar mikroskop üzerinden görsel denetimi altında, yakın hücreye pipet getirmek. </li><li> Yama pipet içinde negatif basınç (H 2 O, 20-40 inç) uygulayın. </li><li> &quot;Osiloskop&quot; penceresinde gigaseal oluşumu izleyin. Test darbe&gt; 5 GΩ pipet direnci artar, azalır ve değeri ile indüklenen akımın genliği, göreceksiniz. Genellikle fe içinde gigaseal formlarıw saniye, ama istikrarlı bir gigaseal elde etmek için 1-2 dakika sürebilir. </li><li> Pipet kapasitans (&quot;C-Hızlı&quot;) giderin. </li><li> 20 cc şırınga kullanarak ek negatif basınç uygulayarak yama membran kırın. </li><li> +30 MV tutma potansiyeli ayarlayın. Olumlu bir tutma potansiyeli kalsiyum-bağımlı Kerek kanal inaktivasyon önlemeye yardımcı olur. </li><li> (&quot;C-yavaş&quot;), hücre zarının kapasitans telafi C-yavaş değeri kaydetmek veya kayıt. </li><li> &quot;R&quot; erişim direnç değerini kontrol edin. Deneylerde, Ar-ler genellikle M 7-20. </li><li> (Şekil 1), Ca 2 + 3 mM Mg 2 + içeren banyo çözüm # 1 0,5 Hz frekans ile gerilim rampaları bir dizi uygulamaya başlayın . Bu çözüm, Mg 2 + Kerek kanalları yoksul geçirgenliği nedeniyle &quot;kaçak&quot; akım ile karşılaştırıldığında Kerek akım yok denecek kadar küçük. Kaydedin ve &quot;kaçak&quot; akımları olarak gelecekteki analizlerde banyo çözüm # 1 kaydedilen mevcut izleri kullanabilirsiniz. -100 MV &quot;kaçak&quot; geçerli mutlak genlik 5 pA eşit veya daha az olmalıdır. Hücreli &quot;sızdıran&quot; ise, yeni bir yama pipet ve yeni bir hücre kullanarak üzerinde deney ve başlangıç ​​durdurun. </li><li> Kerek kanalları (I Ca-Kerek) ile Ca 2 + cari kayıt için perfüzyon vanaları geçiş Ca 2 + ücretsiz 3 mM Mg 2 + içeren banyo çözüm # 1 20 mM Ca 2 + içeren banyo çözüm # 2 (Tablo 1) ile değiştirin. Bu Ca 2 + Kerek kanallar üzerinden akış sağlar ve içe doğru bir akım üretmek. &quot;Osiloskop&quot; penceresinde, içsel ben Ca-Kerek (Şekil 2) giderilmesi gelişimini göreceksiniz. Negatif voltaj seviyelerinde geçerli genliği, Ca 2 +-bağımlı potansiyasyonu Kerek akımı nedeniyle yaklaşık 1 dakika artmaya devam edecek . </li><li> Kerek kanallar aracılığıyla geçici Na + cari kayıt için gerekli olan 5 Hz gerilim rampaları ile stimülasyon frekans artırın. </li><li> 20 mM değiştirin Ca 2 +-perfüzyon vanaları geçiş içeren banyo çözüm # 2 Na +-kalsiyum katyon içeren ücretsiz bir çözüm # 3 (Tablo 1). &quot;Osiloskop&quot; penceresinde, içsel Na giderilmesi büyük genlik geçici bir gelişme göreceksiniz + Akım Kerek kanalları (I Na-Kerek; Şekil 2) ile. </li><li> Bir 20 mM Ca 2 &quot;kaçak&quot; geçerli kaydetmek için deney sonunda 1-5 mcM La 3 + + içeren banyo çözümü içeren # 2 başvurabilir. Ancak, zaman içinde &quot;kaçak&quot; güncel değişiklikler nedeniyle bu yaklaşım yararlı vermedi ve bu deney sonunda daha büyük veya daha küçük olabilir. Ca 2 kaydedilen cari + ücretsiz 3 mM Mg 2 + içeren bir &quot;kaçak&quot; akım olarak deney başında banyo çözüm # 1 almayı tercih </li><li> Daha fazla analiz için kaydedilen akım izleri kaydedin. </li></ol> 7. Veri Analizi <ol><li> Analiz yazılımı içine kaydedilen kayıtları almak. Biz analiz için Darbe programı kullanabilirsiniz. </li><li> Gerilim rampaların başında ve sonunda mevcut genlikleri ölçün. Biz genellikle, -100 mV ve 100 mV Darbe programı &quot;Osiloskop&quot; penceresinde imleçler çiftleri kullanarak mevcut genlikleri ölçer. </li><li> İhracat, mevcut genlikleri değerleri daha fazla analiz ve grafik sunumu için bir grafik programı içine. Biz Kökeni Bilimsel Grafik ve Analiz Yazılımı, sürüm 7 kullanın. </li></ol> 8. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2346/2346fig2.jpg" alt="Şekil 2" /> Şekil 2. Dinlenme insan T hücre Kerek akımları (A), -100 mV (dolu daireler) ve 100 mV (açık daireler) tüm hücre voltaj-klemp yapılandırma kaydedilen Kerek akımların Zaman kursu . Gigaseal oluşumuna önce, hücre Ca 2 ~ 10 dakika için preinkübe + ücretsiz 3 mM Mg 2 + içeren banyo çözümü # 1 - 0.5 mcM thapsigargin içeren Break-sonra, banyo çözümleri sırayla şu şekilde uygulanmıştır: Ca 2 + 3 mM 2 Mg, Ca 2 + içeren banyo çözümü # 2, 20 mM tarafından takip + içeren banyo çözümü # 1 (0 Ca + Mg 3 ), (20 Ca), banyo çözüm # 2 (20 Ca) tarafından takip kalsiyum katyon-banyo çözümü # 3 (DVF) izledi. Hücre Şekil 1&#39;de gösterildiği gibi voltaj rampaları bir dizi ile uyarıldı. Rampa banyo çözüm # 3 (DVF) ve diğer tüm çözümler 0,5 Hz frekans 5 Hz. 20 mM Ca 2 + içeren banyo çözümü # 2 ve Na-Kerek hızlı geçici kalkınma DVF banyo çözüm # 3 uygulamadan sonra yavaş bir gelişme Not I Ca-Kerek . (B), Temsilci akım izleri + ücretsiz 3 mM Mg 2 + içeren banyo çözüm # 1 (Ca 2 gerilim rampaları sırasında kaydedilen&quot;Kaçak&quot;), 20 mM Ca 2 + içeren banyo çözüm # 2 (20 Ca) ve banyo çözümü # 3 (DVF). (C, D), Ca-Kerek (C) ve I Na-Kerek (D) Akım-gerilim ilişkileri voltaj rampaları sırasında 20 mM Ca 2 + içeren banyo kaydedildi akımları &quot;kaçak&quot; mevcut çıkarılarak elde edilen Çözüm # 2 (20 Ca) ve banyo çözümü panelinde gösterilen # 3 (DVF) (B).

<ol>
	<li>Parekh, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+CRAC+channels:+function+in+health+and+disease.">Store-operated CRAC channels: function in health and disease.</a> Nat Rev Drug Discov. 9, 399-410 (2010).</li><li>Feske, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRAC+channelopathies.">CRAC channelopathies.</a> Pflugers Arch. , (2010).</li><li>Lewis, R. S., Cahalan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen-induced+oscillations+of+cytosolic+Ca2++and+transmembrane+Ca2++current+in+human+leukemic+T+cells.">Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.</a> Cell Regul. 1, 99-112 (1989).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+inactivation+of+depletion-activated+calcium+current+(ICRAC)+due+to+local+calcium+feedback.">Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.</a> J Gen Physiol. 105, 209-226 (1995).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+calcium-dependent+inactivation+of+depletion-activated+calcium+current.+Store-dependent+and+-independent+mechanisms.">Slow calcium-dependent inactivation of depletion-activated calcium current. Store-dependent and -independent mechanisms.</a> J Biol Chem. 270, 14445-1451 (1995).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+potentiation+of+store-operated+calcium+channels+in+T+lymphocytes.">Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes.</a> J Gen Physiol. 107, 597-610 (1996).</li><li>Prakriya, M., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+characterization+of+currents+through+store-operated+CRAC+channels+and+Mg2+-inhibited+cation+(MIC)+channels.">Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels.</a> J Gen Physiol. 119, 487-507 (2002).</li><li>Fomina, A. F., Fanger, C. M., Kozak, J. A., Cahalan, . <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+properties+and+regulated+expression+of+Ca(2+)+release-activated+Ca(2+)+(CRAC)+channels+in+human+T+cells.">Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells.</a> J Cell Biol. 150, 1435-1444 (2000).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-Channel Recording. , (1995).</li><li>Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+for+liquid+junction+potentials+in+patch+clamp+experiments.">Correction for liquid junction potentials in patch clamp experiments.</a> Methods Enzymol. 207, 123-1231 (1992).</li></ol>

Insan T hücrelerinin dinlenme Kerek akımların elektrofizyolojik soruşturma, bu hücrelerin endojen Kerek geçerli genliği küçük hücre boyutunu (dinlenme insan T hücre çapı 5-8 mm aralığında) nedeniyle küçük olduğu için zor bir iştir. Burada, periferik kan mononükleer hücreleri izole dinlenme insan T lenfositleri güvenilir Kerek akımları kaydetmek için bir adım-adım prosedürü sunuyoruz. Bu teknoloji bize daha normal ve patolojik immün hücre yanıtlarını doğasını anlamak için dinlenm...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>RosetteSep Human T Cell Enrichment Cocktail</td>
<td>&nbsp;</td>
<td>StemCell Technologies, Vancouver, BC, Canada</td>
<td>15061</td>
<td>&nbsp;</td>
<tr>
<td>RosetteSep Density Medium</td>
<td>&nbsp;</td>
<td>StemCell Technologies </td>
<td>15705</td>
<td>&nbsp;</td>
<tr>
<td>RPMI-in 1640 medium w/glutamine/HEPES</td>
<td>&nbsp;</td>
<td>Fisher, Waltham, MA</td>
<td>SH3025501</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Calf Serum</td>
<td>&nbsp;</td>
<td>Omega Scientific, Tarzana, CA</td>
<td>FB-01</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMAX-I (100X solution)</td>
<td>&nbsp;</td>
<td>Invitrogen, Carlsbad, CA</td>
<td>35050</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 vitamin solution (100X)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>7256</td>
<td>&nbsp;</td>
<tr>
<td>1640 amino acids solution (50X)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R7131</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M7522</td>
<td>&nbsp;</td>
<tr>
<td>Inositol trisphosphate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>19766</td>
<td>&nbsp;</td>
<tr>
<td>BAPTA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A4926</td>
<td>&nbsp;</td>
<tr>
<td>Poly-L-Lysine Hydrobromide</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2636</td>
<td>&nbsp;</td>
<tr>
<td>Lanthanum Chloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>262072</td>
<td>&nbsp;</td>
<tr>
<td>Thapsigargin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>586005</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 Silicon Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning, Midland, MI</td>
<td>3097358-1004</td>
<td>&nbsp;</td>
<tr>
<td>HIPEC R6101 Semiconductor Protective Coating</td>
<td>&nbsp;</td>
<td>Dow Corning, Midland, MI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>63-500 Series High-Performance Vibration Isolation Lab Table</td>
<td>&nbsp;</td>
<td>Technical Manufacturing, Peabody, MA</td>
<td>63-540</td>
<td>&nbsp;</td>
<tr>
<td>EPC 10 patch clamp amplifier with headstage</td>
<td>&nbsp;</td>
<td>HEKA Instruments, Bellmore, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator </td>
<td>&nbsp;</td>
<td>Sutter Instrument, Novato, CA </td>
<td>MP-285</td>
<td>&nbsp;</td>
<tr>
<td>Olympus 1X71 Inverted microscope with 40x oil immersion objective</td>
<td>&nbsp;</td>
<td>Olympus America, Center Valley, PA</td>
<td>1X71</td>
<td>&nbsp;</td>
<tr>
<td>Windows Computer</td>
<td>&nbsp;</td>
<td>Dell </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pulse software</td>
<td>&nbsp;</td>
<td>HEKA Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Origin Scientific Graphing and Analysis Software</td>
<td>&nbsp;</td>
<td>OriginLab, Northampton, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Patch pipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass with filament (O.D.: 1.5mm and I.D.: 1.10mm</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>BF150-110-7.5</td>
<td>&nbsp;</td>
<tr>
<td>Narashige's Microforge</td>
<td>&nbsp;</td>
<td>Tritech Research, Los Angeles, CA</td>
<td>MF-830</td>
<td>&nbsp;</td>
<tr>
<td>Silicon O-rings </td>
<td>&nbsp;</td>
<td>McMASTER-CARR, Santa Fe Springs, CA</td>
<td>111 S70</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips 25 mm </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>12-545-102 25 mm 25CIR.-1</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

whole-cell recording of calcium release-activated calcium (crac) currents in human t lymphocytes

T lenfositleri, tükenmesi Ca 2 + hücre içi Ca 2 + mağaza, aktivasyonu plasmalemmal Ca 2 + kanallarını açar, Kalsiyum Yayın-Aktif Kalsiyum (Kerek) kanalları olarak adlandırılır . Kerek kanal T hücre proliferasyonu ve gen ifadesinin düzenlenmesinde önemli rol oynar. T hücreleri anormal Kerek kanal fonksiyonu ağır kombine immün yetmezlik ve otoimmün hastalıklar 1, 2 bağlantılı olmuştur. Insan T hücreleri Kerek kanal fonksiyonu incelenmesi, normal immün yanıtları düzenleyen yeni moleküler mekanizmaları ortaya çıkarmak ve ilgili insan hastalıklarının nedenleri çözmek olabilir. Membran akımlarının Elektrofizyolojik kayıtları, işlevsel kanal özellikler ve bunların yönetmelik en doğru değerlendirme sağlar. Jurkat T hücreleri, bir insan lösemi T hücre hattı Kerek kanal akımlarının Elektrofizyolojik değerlendirme ilk 3 daha 20 yıl önce yapıldı, ancak, normal insan T hücreleri Kerek akım ölçümleri zorlu bir görev olarak durmaktadır. Kan kaynaklı T lenfositleri Jurkat T hücrelerinin daha boyutu çok daha küçük ve bu nedenle, endojen tam hücreli Kerek akımların genliği çok düşük olduğu gerçeği ile normal T hücreleri Kerek kanal akımları kayıt zorluklar ekleniyor. Burada, biz, biz rutin olarak kaydetmek için kullanabileceğiniz Ca 2 + veya Na + insan T hücrelerinin sağlıklı gönüllülerin periferik kandan izole edilen dinlenme Kerek kanalları üzerinden akımlar. Bir adım-adım prosedürü vermek Burada anlatılan yöntemi, Jurkat T hücreleri Kerek akımları kayıt ve insan T hücrelerinin 4-8 aktive için kullanılan usuller kabul edilmiştir .

Watch this Scientific Journal Video about Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes at JoVE.com

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

Biz, Kalsiyum-Film Aktive Kalsiyum tam hücreli yama kelepçe kayıt (Kerek) periferik kan mononükleer hücre-kökenli insan T lenfositleri akımları için bir adım-adım protokolü sağlar.

İnsan T lenfositleri, Kalsiyum-Film Aktive Kalsiyum Tüm Hücre Kayıt (Kerek) Akımlar

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

whole-cell-recording-calcium-release-activated-calcium-crac-currents

Research

JoVE Journal

Biology

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

16.0K Görüntüleme.  University of California, Davis. Biz, Kalsiyum-Film Aktive Kalsiyum tam hücreli yama kelepçe kayıt (Kerek) periferik kan mononükleer hücre-kökenli insan T lenfositleri akımları için bir adım-adım protokolü sağlar.

Video: Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Kalsiyum Göstergeleri ile yükleme Drosophila Sinir Terminalleri

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Biyoloji

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

Katman In Vivo 2-Foton Kalsiyum Görüntüleme Fare 2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

Işık tam hücreli Kayıtlar Retina Dilim Uyarıcı Synaptic Akımlar Uyarılmış

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Astrositler Plazma Membran ve Küresel Hücre içi kalsiyum Dynamics civarında Ölçüm

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Apoptozis sırasında Mitokondrial Kalsiyum Düzeyleri Dinamik değişiklikler izlenmesi Genetiği Kodlanmış Kalsiyum Sensor Kullanımı

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Hızlı Kalsiyum Dekapanları kardiyomiyositlerde Ölçme

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

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 ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Sitometrisi ile Böbrek Hücrelerine sitozolik kalsiyum Ölçümleri

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Floresan Biyoalgılayıcı kullanarak böcek besleme sırasında gerçek zamanlı Vivo kayıt Arabidopsis kalsiyum sinyallerin

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Fare Pankreas Adacık Hücrelerinin Alt Popülasyonlarında Görüntüleme Kalsiyum Dinamiği

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...