Yazarlar ifşa gerek yok.

1. INS-1E hücreleri: bakım ve kaplama
<ol><li>INS-1E hücreleri oksijen 37 ˚C/5% CO2 kuluçka ve kültürlü RPMI 1640 orta ile %5 (v/v) ısı inaktive fetal sığır serum ile 2 mM L-glutamin, 10 mM HEPES, 1 mM sodyum pyruvate, 100 U/mL penisilin/streptomisin takıma korumak çözüm, 50 µM β-mercaptoethanol. 10 mL tam RPMI 1640 orta (plaka), başına hücrelerde kültür % 80-90 izdiham ulaşana kadar ne zaman onlar trypsinized ve pasajlı veya insülin salgısı tahlil için kullanılan.</li>
	<li>
1. gün: Medya Aspire edin ve hücreleri bir kez 5 mL de önceden ısıtılmış PBS ile yıkayın. Tripsin (% 0,025) 0.5 mL 1:1 0.5 ml hücreleri trypsinize ve 3-4 dk 37 ˚C az için kuluçkaya için PBS seyreltilmiş ekleyin. Tripsin 15 mL santrifüj tüpü 9 mL tam medya ve transfer hücreleri ekleyerek pipetting tarafından devre dışı bırakın.</li>
	<li>Hücreleri tarafından Santrifüjü cips ve hücre Pelet yaklaşık 5 mL taze medyada yeniden askıya alma.</li>
	<li>Yeniden askıya alınan hücre 10 µL alıp Trypan mavi hayati boya için hücre canlılığı kontrol etmek için 10 µL ile karıştırın. Bu karışımdan bir hemasitometre kullanarak 10 µL canlı ve ölü hücreleri saymak. Canlılığı düzeyi %90 üzerinde olmalıdır. Taze medya mL başına 1 milyon hücrelere yeniden askıya alınmış hücrelerde sulandırmak.</li>
	<li>Poli-L-lizin bir kuyu başına tohum 0.5 mL INS-1E hücre 24-şey plaka, 500.000 hücreler/iyi bir yoğunluğu ön kaplamalı.</li>
	<li>
Gün 2: Medya 18-24 h kaldırmak sonra kaplama ve 500 µL/iyi taze RPMI 1640 medya ekleyin. Hücreler hücreleri tamamen önceden passaging adımından kurtarmak izin vermek başka bir 24 h için kuluçkaya. Bu ek süre hücre doku kültürü plaka üzerinde yaymak için izin verir.</li>
</ol>2. insülin salgısı tahlil (3. gün)
<ol><li>KRB arabellek hazırlamak: 132,2 mM NaCl, 3.6 mM KCl, 5 mM NaHCO3, 0,5 mM NaH2PO4, 0,5 mM MgCl2, 1.5 mM CaCl2ve 0,001 g/mL sığır serum albumin (BSA), pH 7.4.</li>
	<li>Medya hücreleri Aspire edin ve iki kez Önceden ısıtılmış PBS ile yıkayın.</li>
	<li>Glukoz açlık adım için 450 µL/iyi KRB (içeren BSA) 1 h için glikoz olmadan 37 ˚C/5% CO2' de ekleyin.</li>
	<li>Glukoz açlık adım sırasında ilaç (lar) 200 mM içeren KRB içinde seri dilutions glikoz hazırlama (10 x konsantrasyon).<ol>
<li>10 x 200 mM glikoz (de 10 final tahlil glikoz konsantrasyonu x) ile takıma KRB son konsantrasyon uyuşturucu hazırlayın. Eğer uyuşturucu hisse senedi (ek 10 artı glikoz x) olduğunu DMSO, DMSO yüzde tahlil (ideal olarak son yüzdesi % 0,1 DMSO daha az) tutarlı tutulur emin olun.</li>
		<li>Dopamin ve quinpirole tedavisi için 100 100 µM bir son tahlil ilaç konsantrasyonu aralığını kullanmak hiç uyuşturucu içeren doz yanıtı son nokta ile (yüksekten en düşük konsantrasyon), pM. Bromokriptin için bir son tahlil konsantrasyon aralığını 10 µM ile 22 saatleri, ilaçsız kontrolü doz yanıt son nokta ile kullanmak.</li>
	</ol>
</li>
	<li>Glukoz açlık sonra uyuşturucu seri dilutions bir doz yanıt üretmek için tahlil için ekleyin.</li>
	<li>Her seri seyreltme 50 µL/kuyu karşılık gelen wells (Toplam tahlil birim 500 µL) ekleyin.</li>
	<li>37 ˚C/5% CO, 90 dk için ilgili uyuşturucu seri dilutions (huzurunda 20 mM glikoz) içeren hücreleri glikoz stimülasyon adım için kuluçkaya2. Bir denetim wells içerir: (1) uyarılması ile 20 mM glikoz yokluğunda yalnız herhangi bir ek ilaç ve (2) hücreleri, ne uyuşturucu ne de (salgı Bazal oranında sağlayan) glikoz ile uyarılır.</li>
	<li>Stimülasyon adımdan sonra supernatants (doğrudan kullanım veya 4 ˚C mağazasında) dikkatli bir şekilde çıkarın. Not: Bir ek 5 dk nazik Santrifüjü adım (600 x g, 1 dk) bu noktada herhangi bir kalan hücreleri tahlil supernatants kaldırmak için tanıttı.</li>
</ol>3. ölçü insülin salgılanması için HTRF
<ol><li>Tahlil supernatants 1:10 KRB (olmadan BSA), tercihen temiz 96-şey levha oranında seyreltin.</li>
	<li>İnsülin standart eğri HTRF insülin tahlil (tablo 1) için hazır olun.</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Standart stok çözüm 500 ng/ml</td>
			<td>Seri dilutions</td>
			<td>[İnsülin] ng/ml çalışma</td>
		</tr>
<tr>
<td>STD 7</td>
			<td>30 µl stok + 140 µl KRB</td>
			<td>150</td>
		</tr>
<tr>
<td>STD 6</td>
			<td>30 µl STD 7 + 45 µl KRB</td>
			<td>60</td>
		</tr>
<tr>
<td>STD 5</td>
			<td>30 µl STD 6 + 45 µl KRB</td>
			<td>24</td>
		</tr>
<tr>
<td>STD 4</td>
			<td>30 µl STD 5 + 45 µl KRB</td>
			<td>9.6</td>
		</tr>
<tr>
<td>STD 3</td>
			<td>30 µl STD 4 + 45 µl KRB</td>
			<td>3.84</td>
		</tr>
<tr>
<td>STD 2</td>
			<td>30 µl STD 3 + 45 µl KRB</td>
			<td>1,54</td>
		</tr>
<tr>
<td>STD 1</td>
			<td>30 µl STD 2 + 45 µl KRB</td>
			<td>0,61</td>
		</tr>
<tr>
<td>STD 0</td>
			<td>45 µl KRB</td>
			<td>0</td>
		</tr>
<tr>
<td colspan="3">Not: STD hisse senedi 500 ng/mL'dir</td>
		</tr>
</tbody></table>Tablo 1. İnsülin standart eğri yapmak için seri dilutions.
<ol start="3"><li>Standart eğri örnekleri ve seyreltilmiş tahlil supernatants HTRF plakasına ekleyin. HTRF tarafından salgılanan insülin ölçümü bir 96-kuyu veya yeniden ayarlanması için tahlil birim olan göz önünde bulundurarak bir 384-şey plaka biçim yapılabilir. Kullanım 10 µL/iyi örnek 96-şey beyaz yarı-alan plakaları veya 5 µL/iyi 384-şey beyaz bir düşük hacimli, yuvarlak alt plaka ( Tablo malzemelerigörmek).</li>
	<li>Antikor mix algılama arabelleği hazırlamak ( Tablo malzemelerigörmek) 1:2 donör (cryptate) içinde / alıcısı (XL-665) oranı. Not: Daha fazla HTRF tahlil ilgili belirli ayrıntıları üreticisinden mevcuttur.</li>
	<li>30 µL/iyi (için 96 iyi-plaka tahlil biçimi) veya 15 µL/iyi (384-şey plaka tahlil biçimi için) tahlil için antikor karışımı ekleyin.</li>
	<li>Plaka mühür ve oda sıcaklığında kuluçkaya.</li>
	<li>Plaka plaka okuyucu ve uygun HTRF optik modülü kullanarak antikorlar ile 2 h, 4 h ve/veya gece kuluçka sonra okumak (337 665 620 nm) ( Tablo malzeme ve üretici yönergelerine bakın). 60 µs entegrasyon başlangıç ayarlamak ve entegrasyon anda 400 µs. kullanım 200 iyi yanıp söner. Not: Bu parametreler belirli bizim okuyucu kullan seçeneğine göre. Okuma, 620 nm ve 665 nm farklı enstrümanlar arasında farklı olabilir. 665/620 oranı kullanmanızı öneririz nedenlerinden biridir. Bu oran hesaplama, herhangi bir potansiyel farkları okuyucudan okuyucuya normalleştirilmiş ve HTRF ölçmek için kullanılan alet bakılmaksızın tutarlı değerleri sağlar.</li>
</ol>4. veri analizi ve normalleştirme
<ol><li>Tahlil kuyu ratiometric ekstrapolasyon yoluyla insülin konsantrasyonları hesaplamak Floresans okuma (665 nm/620 nm) için ikinci sırada ikinci dereceden bir polinom eğrisi (Şekil 2).</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57531/57531fig2.jpg"> Resim 2 : İnsülin standart eğri. Bilinen konsantrasyonları stokunun insan insülin, insülin standart eğri oluşturmak için kullanılmıştır. Elde edilen HTRF oranları (665 nm / 620 nm) insülin konsantrasyonları karşı çizildi. Verileri ikinci sırada ikinci dereceden bir polinom eğrisi için en uygun olduğunu (R2 0.99996 =). Bu temsili bir standart eğri var. Hata çubukları SEM = <a href="/files/ftp_upload/57531/57531fig2large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<ol start="2"><li>Ng/mL salgılanan insülin olarak ekstrapole verilerle (ligand konsantrasyonu artan karşılık salgılanan insülin) % maksimum insülin salgısı tahlil wells (20 mM glikoz yalnız koşulu) ortalama değerine normalleştirmek.</li>
	<li>Eğrisi hastalık nöbeti (R2) tek bir deneme intraplate varyasyon hesaplamak için kullanın. Deney içinde uygun eğrisi için ortalama, standart hata hesaplama sağlayan intra deneysel çoğaltmaları bireysel R2 değerleri türetilen.</li>
	<li>İnterplate varyasyon belirlemek için en az üç bireysel deney verileri toplu eğrisi R2 değeri için ortalama, standart hata hesaplamak için kullanın.</li>
</ol>

<ol>
	<li>Taniguchi, C. M., Emanuelli, B., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+nodes+in+signalling+pathways:+Insights+into+insulin+action.">Critical nodes in signalling pathways: Insights into insulin action.</a> Nat Rev Mol Cell Biol. 7 (2), 85-96 (2006).</li><li>Vetere, A., Choudhary, A., Burns, S. M., Wagner, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+pancreatic+beta-cell+to+treat+diabetes.">Targeting the pancreatic beta-cell to treat diabetes.</a> Nat Rev Drug Discov. 13 (4), 278-289 (2014).</li><li>Despres, J. P., Lemieux, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+obesity+and+metabolic+syndrome.">Abdominal obesity and metabolic syndrome.</a> Nature. 444 (7121), 881-887 (2006).</li><li>Kahn, S. E., Hull, R. L., Utzschneider, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+linking+obesity+to+insulin+resistance+and+type+2+diabetes.">Mechanisms linking obesity to insulin resistance and type 2 diabetes.</a> Nature. 444 (7121), 840-846 (2006).</li><li>Barg, S. <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+exocytosis+in+insulin-secreting+B-cells+and+glucagon-secreting+A-cells.">Mechanisms of exocytosis in insulin-secreting B-cells and glucagon-secreting A-cells.</a> Pharmacol Toxicol. 92 (1), 3-13 (2003).</li><li>Braun, M., Ramracheya, R., Rorsman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+regulation+of+insulin+secretion.">Autocrine regulation of insulin secretion.</a> Diabetes Obes Metab. 14 Suppl 3, 143-151 (2012).</li><li>Farino, Z. 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=Development+of+a+rapid+insulin+assay+by+homogenous+time-resolved+fluorescence.">Development of a rapid insulin assay by homogenous time-resolved fluorescence.</a> PLoS One. 11 (2), e0148684 (2016).</li><li>Simpson, N., 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=Dopamine-mediated+autocrine+inhibitory+circuit+regulating+human+insulin+secretion+in+vitro.">Dopamine-mediated autocrine inhibitory circuit regulating human insulin secretion in vitro.</a> Mol Endocrinol. 26 (10), 1757-1772 (2012).</li><li>Heyduk, E., Moxley, M. M., Salvatori, A., Corbett, J. A., Heyduk, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneous+insulin+and+C-Peptide+sensors+for+rapid+assessment+of+insulin+and+C-peptide+secretion+by+the+islets.">Homogeneous insulin and C-Peptide sensors for rapid assessment of insulin and C-peptide secretion by the islets.</a> Diabetes. 59 (10), 2360-2365 (2010).</li><li>Heyduk, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+protein+conformational+changes+by+FRET/LRET.">Measuring protein conformational changes by FRET/LRET.</a> Curr Opin Biotechnol. 13 (4), 292-296 (2002).</li><li>Degorce, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTRF((R)):+Pioneering+technology+for+high-throughput+screening.">HTRF((R)): Pioneering technology for high-throughput screening.</a> Expert Opin Drug Discov. 1 (7), 753-764 (2006).</li><li>Degorce, 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=HTRF:+A+technology+tailored+for+drug+discovery+-+a+review+of+theoretical+aspects+and+recent+applications.">HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications.</a> Curr Chem Genomics. 3, 22-32 (2009).</li><li>Mathis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTRF(R)+Technology.">HTRF(R) Technology.</a> J Biomol Screen. 4 (6), 309-314 (1999).</li><li>Daijo, J. E., Sportsman, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-resolved+fluorescence+immunoassay+for+insulin+in+rodent+plasma.">A time-resolved fluorescence immunoassay for insulin in rodent plasma.</a> J Pharm Biomed Anal. 19 (3-4), 335-342 (1999).</li><li>Merglen, A., 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=Glucose+sensitivity+and+metabolism-secretion+coupling+studied+during+two-year+continuous+culture+in+INS-1E+insulinoma+cells.">Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells.</a> Endocrinology. 145 (2), 667-678 (2004).</li><li>Ustione, A., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+synthesis+and+D3+receptor+activation+in+pancreatic+beta-cells+regulates+insulin+secretion+and+intracellular+[Ca(2+)]+oscillations.">Dopamine synthesis and D3 receptor activation in pancreatic beta-cells regulates insulin secretion and intracellular [Ca(2+)] oscillations.</a> Mol Endocrinol. 26 (11), 1928-1940 (2012).</li><li>Ustione, A., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+introduction+to+multiphoton+microscopy.">A simple introduction to multiphoton microscopy.</a> J Microsc. 243 (3), 221-226 (2011).</li><li>Ustione, A., Piston, D. W., Harris, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+Dopaminergic+regulation+of+insulin+secretion+from+the+pancreatic+islet.">Minireview: Dopaminergic regulation of insulin secretion from the pancreatic islet.</a> Mol Endocrinol. 27 (8), 1198-1207 (2013).</li><li>Rubi, B., 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=Dopamine+D2-like+receptors+are+expressed+in+pancreatic+beta+cells+and+mediate+inhibition+of+insulin+secretion.">Dopamine D2-like receptors are expressed in pancreatic beta cells and mediate inhibition of insulin secretion.</a> J Biol Chem. 280 (44), 36824-36832 (2005).</li><li>Garcia-Tornadu, I., 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=Disruption+of+the+dopamine+d2+receptor+impairs+insulin+secretion+and+causes+glucose+intolerance.">Disruption of the dopamine d2 receptor impairs insulin secretion and causes glucose intolerance.</a> Endocrinology. 151 (4), 1441-1450 (2010).</li><li>Garcia-Tornadu, I., 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=New+insights+into+the+endocrine+and+metabolic+roles+of+dopamine+D2+receptors+gained+from+the+Drd2+mouse.">New insights into the endocrine and metabolic roles of dopamine D2 receptors gained from the Drd2 mouse.</a> Neuroendocrinology. 92 (4), 207-214 (2010).</li><li>Lopez Vicchi, 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=Dopaminergic+drugs+in+type+2+diabetes+and+glucose+homeostasis.">Dopaminergic drugs in type 2 diabetes and glucose homeostasis.</a> Pharmacol Res. 109, 74-80 (2016).</li><li>Kalra, S., Kalra, B., Agrawal, N., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine:+The+forgotten+felon+in+type+2+diabetes.">Dopamine: The forgotten felon in type 2 diabetes.</a> Recent Pat Endocr Metab Immune Drug Discov. 5 (1), 61-65 (2011).</li><li>Mahajan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromocriptine+mesylate:+FDA-approved+novel+treatment+for+type-2+diabetes.">Bromocriptine mesylate: FDA-approved novel treatment for type-2 diabetes.</a> Indian J Pharmacol. 41 (4), 197-198 (2009).</li><li>Caicedo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+and+autocrine+interactions+in+the+human+islet:+more+than+meets+the+eye.">Paracrine and autocrine interactions in the human islet: more than meets the eye.</a> Semin Cell Dev Biol. 24 (1), 11-21 (2013).</li><li>Ballon, J. S., Pajvani, U., Freyberg, Z., Leibel, R. L., Lieberman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathophysiology+of+metabolic+effects+of+antipsychotic+medications.">Molecular pathophysiology of metabolic effects of antipsychotic medications.</a> Trends Endocrinol Metab. , (2014).</li><li>Freyberg, Z., Aslanoglou, D., Shah, R., Ballon, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+and+antipsychotic+drug-induced+metabolic+dysfunction+in+schizophrenia.">Intrinsic and antipsychotic drug-induced metabolic dysfunction in schizophrenia.</a> Front Neurosci. 11, 432 (2017).</li><li>de Leeuw van Weenen, E. 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=The+dopamine+receptor+D2+agonist+bromocriptine+inhibits+glucose-stimulated+insulin+secretion+by+direct+activation+of+the+alpha2-adrenergic+receptors+in+beta+cells.">The dopamine receptor D2 agonist bromocriptine inhibits glucose-stimulated insulin secretion by direct activation of the alpha2-adrenergic receptors in beta cells.</a> Biochem Pharmacol. 79 (12), 1827-1836 (2010).</li></ol>

Biz Nicolas Pierre (Cisbio BİYOANALİZLER) için faydalı tavsiyeler ve Dr. Pierre Maechler (Cenevre Üniversitesi) cömertçe INS-1E hücreleri verdiğiniz için teşekkür ederiz. Bu eser Savunma Bakanlığı (grant PR141292 Z.F. için) ve John F. ve Nancy A. Emmerling Fonu (için Z.F.) Pittsburgh Vakfın fon tarafından desteklenmiştir.

Burada açıklanan HTRF insülin tahlil insülin salgılanması kültürlü hücre tabanlı sistemden ölçmek için hızlı, verimli bir sistem sunmaktadır. En önemli avantajları arasında bu tahlil yüksek sinyal gürültü oranı nedeniyle düşük arka plan sinyal sunmaktadır. Buna ek olarak, biz HTRF sinyal uzun bir süre için kararlı olduğunu doğruladı (> 24 h)7. Yine de, insülin bağlama monoklonal antikorlar, hızlı bir şekilde tahlil için ek sonra bağlama doygunluk ulaşmak ...

Enerji metabolizmasının büyük bir anabolik hormon tarafından insülin ince ayar. İnsülin sentezlenmiş ve yanıt olarak artan hücre dışı şekeri pankreas beta hücreleri tarafından yayımlanan. Yayımlanan insülin glukoz alımını insülin duyarlı dokular1,2tarafından tetikler. Fizyolojik açıdan, bu glikoz konsantrasyonu glukoz alımını düzenleyen insülin salgılanmasını tarafından takip bir yemekten sonra yükselmesine bağlı olduğu. İnsülin direnci ile sonuçlanan metabolik bozukluklar glikoz homeostazı bozuklukları neden olabiliyor ve sonuçta, tip 2 diyabet2,3,4başlangıcı.
Her ne kadar insülin salgılanması kapsamlı eğitimi aldı, onun düzenleyici mekanizmaları kötü anlaşılır kalır. Soruşturmanın kritik bir alan kimliği roman modülatörler insülin salgılanması beta hücreleri5,6,7,8tarafından olmuştur. Bu çalışmalar glikoz stimülasyon ve insülin salgılanması kaplin ilişkisi daha iyi anlaşılmasını gerektirir. Bu nedenle, doğru bir şekilde izlemek ve glikoz uyarılmış insülin salgılanması (GSIS) düzeylerini ölçmek için yetenek gerekli olmuştur. Bugüne kadar ancak, sınırlı sayıda yöntemleri GSIS insülin salgılayan hücre satırları ve/veya pankreas adacıkları kullanarak miktar izin vermek mevcut idi. Radioimmunoassay (RIA), hangi Radyoizotop öğesini insülin ve antikorlar biridir. Bu yaklaşımın ana sınırlamaları nedeniyle işleme ve bertaraf radyoaktif malzemelerin güvenlik sorunları içerir. Ayrıca, bu emek yoğun, birden çok uzun çamaşır ve kuluçka adımları içeren bir yöntemdir. Enzim bağlı immunosorbent assay (ELISA) insülin algılama için antikorlar kullanan başka bir pahalı ve emek yoğun bir yaklaşımdır. Varyasyon antikor benzeşim ve insülin tanıma verimliliğini faktörler bu yöntemin sınırlama var ve tekrarlanabilirlik sonuçları etkileyebilir. Ne ELISA ne de RIA yüksek üretilen iş deneyler için tasarlanmıştır. AlphaScreen algılama ve insülin salgılanması düzeylerini ölçmek için kullanılan homojen bir tahlil olduğunu. AlphaScreen teknoloji ortam oksijen dönüşüm Kemiluminesan üretimi kaynaklanan chemiluminescent türleri ile tepki verebilir bir heyecanlı oksijen singlet durumuna dayanır. Tahlil homojen olduğundan, RIA ve ELISA ile ilgili çamaşır adımlardan ortadan kalkar. Ancak, sinyal tepki doğası gereği istikrarsızlık tahlil okuma etkileyebilecek bir sınırlayıcı faktördür. (TR-KISKAÇ, Heyduk ve meslektaşları9tarafından geliştirilen başka bir homojen insülin molekül üzerinde farklı epitopları için iki ayrı antikorların bağlama göre insülin ölçüm yaklaşımdır. Antikorlar her kimyasal olarak bağlantılı çift telli DNA'sı ile kısa tamamlayıcı tek iplikçikli DNA çıkıntılar vardır. İnsülin antikorları bağlama onları bir araya getirir ve bir çift telli DNA dubleks için yol açar. Her antikor da ilgili bir donör veya alıcısı fluorophore ile ilişkilidir ve DNA çift yönlü Derneği Förster rezonans enerji transferi (FRET) oluşturmak için bu fluorophores buluşturuyor. TR-KISKAÇ, bir potansiyel sınırlama ancak, perde ile aittir. Arka plan Floresans FRET reaksiyon sırasında hızla dağılmaya yetersizlik arka plan Floresans nispeten yüksek düzeyde ve düşük sinyal gürültü oranı tahlil içinde neden olabilir. Bu nedenle, bir ihtiyaç GSIS bir yüksek-den geçerek şekilde miktarının için güvenilir, sağlam ve uygun maliyetli bir tahlil için devam eder.
Biyofizik son gelişmeler bir homojen zaman çözüldü floresan enerji rezonans transferi (HTRF) dayalı tahlil gelişiminde sonuçlandı. Enerji aktarım içinde özellikle, tahlil daha doğru bir şekilde donör ve alıcısı arasındaki enerji transferi ışınımsal olmayan ışıma enerji rezonans transferi (LRET)10 HTRF dayanan FRET dayalı olarak açıklanan olabilir tür11,12,13. Bu ayrım bir floresan zamanlama beri önemli, ya da perdeleme aynı türleri FRET ve LRET için kullanılan temel perde etkileşim Şoklama LRET için daha çok farklı olsa da. Ayrıca, nadir toprak lanthanide cryptate kullanımı Evropyum gibi maddeler ve birleşimler veya uzun floresan yarı-hayat12,14HTRF terbium cryptate üretir. Bu belirli bir gecikme süresiyle (µsn) donör uyarma ve alıcısı (Yani, zaman çözüldü tahlil) emisyon ölçümü arasında giriş benzersiz avantajı sunuyor. Bu gecikme süresini alıcısı emisyon Floresans ölçüm önce dağılımı arka plan floresan için yeterli zaman sağlar. Sonuç olarak, okuma non-spesifik Floresans ücretsiz ve böylece, bir yüksek sinyal gürültü oranı elde edilir mi (şekil 1). Ayrıca, HTRF homojen doğası çok tahlil yapmak ilişkisiz türler ELISA veya RIA tabanlı yöntemleri daha daha fazla hızlı yıkama adımları yıkamak için gereksinimini ortadan kaldırır.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57531/57531fig1.jpg"> Şekil 1: HTRF insülin algılama için mekanizmasının şematik. İki bağımsız olarak oluşturulan monoklonal antikorlar özellikle tanımak ve insülin ayrı sitelerde bağlayın. Bu antikorlar Evropyum cryptate donör veya XL665 alıcısı için Birleşik. Uyarma 337 nm sonuçlara emisyon 620, içinde bağış nm. Elde edilen enerji transferi bir daha uzun dalga boyu, 665 yayılmasını sağlamak XL665 neden olan nm. <a href="/files/ftp_upload/57531/57531fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
Burada, detaylı bir protokol sağlar GSIS INS-1E hücrelerden düzeylerini belirlemek için HTRF dayalı bir yaklaşım kullanmak için insülinoma hücre satır15sıçan bir iyi kurulmuş insülin salgılayan beta hücre kaynaklı. Buna ek olarak, bu tahlil insülin salgılanması moleküler düzenleyiciler farmakolojik profili tanımlamak için kullanılabilir. Dopamin D2incelemek için bu HTRF tabanlı insülin testin uyguladığımız-reseptör düzenleme GSIS, hangi tarihlerde. Artan çalışmalar nörotransmitter dopamin GSIS8,16,17,18,19,20, önemli bir düzenleyicisidir indirdik 21 , 22. dopamin bir negatif otokrin/parakrin şekilde dopamin D2eylemler yoluyla GSIS etkiler-tarihlerde beta pankreas hücreleri8 yüzeyde ifade reseptörleri (D2, D3, D4 reseptörleri) , 16 , 19. bu tahlil kullanarak, biz GSIS olumsuz bir düzenleyici olarak dopamin'ın rolü onaylamak ve gösteren dopamin D2-reseptör agonistleri bromokriptin ve quinpirole da GSIS azaltmak gibi.

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			<td height="42" style="height:42px;width:216px;">96-well white half area plate</td>
			<td style="width:104px;">Greiner Bio-One</td>
			<td style="width:139px;">82050-042</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">384-well white low-volume, round-bottom plate</td>
			<td style="width:104px;">Corning</td>
			<td>15100157</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insulin High Range Kit</td>
			<td style="width:104px;">Cisbio Bioassays</td>
			<td style="width:139px;">62IN1PEH&#160;</td>
			<td>10,000 test HTRF-based insulin assay kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PHERAstar FSX plate reader</td>
			<td style="width:104px;">BMG Labtech</td>
			<td>FSX</td>
			<td>Plate reader adapted for HTRF-based assay readings</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plate sealers</td>
			<td style="width:104px;">Fisher Scientific</td>
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			<td>To seal plate while antibodies incubate</td>
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			<td style="width:104px;">Hausser Scientific</td>
			<td style="width:139px;">3100</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">RPMI Medium 1640 (1x)</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">11875-093</td>
			<td>[+] L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin 0.05%&#160;</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">25-052-CI</td>
			<td>0.53 mM EDTA, (-) sodium bicarbonate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DPBS (Dulbecco&#39;s phosphate buffered saline)</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">21-031-CV</td>
			<td>Without calcium and magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
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			<td style="width:139px;">35-010-CV</td>
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			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:104px;">Gibco&#160;</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium pyruvate</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">11360070</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin/Streptomycin solution 100x</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">30-002 CT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">M1348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue Stain (0.4%)</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dopamine hydrochloride</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">8502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bromocriptine mesylate</td>
			<td style="width:104px;">Tocris</td>
			<td style="width:139px;">427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">(-)-Quinpirole hydrochloride</td>
			<td style="width:104px;">Tocris</td>
			<td style="width:139px;">1061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:104px;">Calbiochem</td>
			<td style="width:139px;">12659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poly-L-Lysine</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">P4832</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO (Dimethyl sulfoxide)</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">276855</td>
			<td></td>
		</tr>
	</tbody>
</table>

homogeneous time-resolved f&#246;rster resonance energy transfer-based assay for detection of insulin secretion

İnsülin salgılanması tespiti düzenlenmiş salgılanmasını de çalışmalar olduğu gibi metabolizma elucidating mekanizmaları için önemlidir. Çok sayıda insülin deneyleri yıllardır varmışsın, homojen zaman çözüldü Förster rezonans enerji transferi (HTRF) teknolojisi son gelişiyle önemli ölçüde bu ölçümler basitleştirdi. Bu hızlı, uygun maliyetli, tekrarlanabilir ve güçlü optik tahlil zaman çözüldü Förster enerji rezonans Transfer kolaylaştırır uzun süreli emisyon ile parlak fluorophores için Birleşik antikorları üzerine bağımlı olduğunu. Ayrıca, HTRF insülin algılama yüksek üretilen iş tarama testleri geliştirme için mükellef olduğunu. Burada HTRF insülin salgılanması INS-1E hücrelerde, bir sıçan insülinoma türetilmiş hücre kültürünü algılamak için kullanır. Bu bize Bazal düzeyde insülin ve glikoz stimülasyon cevaben yaptıkları değişiklikleri tahmin etmek izin verir. Buna ek olarak, bu insülin algılama sistemi dopamin rol glikoz uyarılmış insülin salgılanması (GSIS) negatif bir düzenleyici olarak onaylamak için kullanın. Benzer bir şekilde, diğer dopamin D2-reseptör agonistleri, quinpirole ve bromokriptin, GSIS bir konsantrasyon bağımlı şekilde azaltmak gibi. Sonuçlarımız HTRF insülin tahlil biçiminin GSIS ve farmakolojik kendi profilleri çok sayıda uyuşturucu rolünü belirlemede yardımcı programı vurgulayın.

Biz bizim insülin HTRF tahlil önceden tanımlanmış konsantrasyonları (Şekil 2) saf insan insülin Standartlar kullanılarak bir insülin standart eğri üreterek geçerliliği. Üretimi ratiometric floresan okuma ulaşmak için bize izin verilen standart eğri ve bu nedenle ilaç tedavileri (Şekil 2) yanıt olarak salgılanan insülin düzeylerini belirlemek. Intraplate değişim eğrisi montaj için en az (R...

Watch this Scientific Journal Video about Homogeneous Time-resolved FÃƒÂ¶rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion at JoVE.com

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Burada, homojen mevcut zaman hücrelerden salgılanan insülin hızlı algılama için etkili bir yöntem olarak FRET (HTRF) çözüldü.

Homojen zaman çözüldü Förster rezonans enerji transferi tabanlı tahlil insülin salgılanması algılanması için

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

homogeneous-time-resolved-frster-resonance-energy-transfer-based

Research

JoVE Journal

Medicine

9.1K Görüntüleme.  University of Pittsburgh. Burada, homojen mevcut zaman hücrelerden salgılanan insülin hızlı algılama için etkili bir yöntem olarak FRET (HTRF) çözüldü.

Video: Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Kanserler Üretim ve reaktif oksijen türlerinin (ROS Algılama)

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Tıp

Improving IV Insulin Administration in a Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4 The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5 It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6 Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p&lt;0.001). From this pivotal trial and two others,7,8 Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia. 
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG &lt; 40 mg/dL), clinical hypoglycemia (BG &lt; 70 mg/dL), and hyperglycemia (BG &gt; 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG &lt; 70 mg/dL) with the use of the computer glucose management system was well under 1%.

When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (&lt;40 mg/dL), clinical hypoglycemia (&lt;70 mg/dL), and hyperglycemia (&gt;180 mg/dL) also can be observed.

Bir Topluluk Hastanesi IV insülin uygulaması Geliştirilmesi

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Hafif Endometriozis İntraoperatif Algılama: Periton Bütünlüğü Kaybı ile İlişkili Pelvik Ağrı Tespiti ve Tedavisi İçin Yeni Bir Paradigma

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Kanser Tedavisi İzleme Cerenkov Lüminesans Görüntüleme (CLI)

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.

Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method&#x2014;adapted from a coculture model of synaptogenesis&#x2014;for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.

Pankreas Beta Hücreleri ile insülin salgılanması Etkileyen Ekstrasellüler Protein etkileşimleri kokültürü Analizi

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

Intravital mikroskobu ve kontrast-artırılmış Ultrasonografi fare Hindlimb, insülin kaynaklı vazodilatasyon ve kas perfüzyon çalışmaya kombine

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Cardiac Magnetic Resonance Imaging at 7 Tesla

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher magnetic field strengths and potentially provides improved signal contrast and spatial resolution. While promising results have been achieved, ultra-high field CMR is challenging due to energy deposition constraints and physical phenomena such as transmission field non-uniformities and magnetic field inhomogeneities. In addition, the magneto-hydrodynamic effect renders the synchronization of the data acquisition with the cardiac motion difficult. The challenges are currently addressed by explorations into novel magnetic resonance technology. If all impediments can be overcome, ultra-high field CMR may generate new opportunities for functional CMR, myocardial tissue characterization, microstructure imaging or metabolic imaging. Recognizing this potential, we show that multi-channel radio frequency (RF) coil technology tailored for CMR at 7 Tesla together with higher order B0 shimming and a backup signal for cardiac triggering facilitates high fidelity functional CMR. With the proposed setup, cardiac chamber quantification can be accomplished in examination times similar to those achieved at lower field strengths. To share this experience and to support the dissemination of this expertise, this work describes our setup and protocol tailored for functional CMR at 7 Tesla.

The sensitivity gain inherent to ultrahigh field magnetic resonance holds promise for high spatial resolution imaging of the heart. Here, we describe a protocol customized for functional cardiovascular magnetic resonance (CMR) at 7 Tesla using an advanced multi-channel radio-frequency coil, magnetic field shimming and a triggering concept.

7 Tesla, kardiyak manyetik rezonans görüntüleme

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Hipotalamik insülin sinyal fare beyin içine sürekli uyuşturucu infüzyon sistemi ile periferik glikoz intoleransı için eğitim

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

Eleştirel hasta hastalarda tam kan endotoksemi tespiti için endotoksin aktivite assay

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

Gut hormonu salgılanması izole kullanarak temel mekanizmaları fare bağırsağı periosteum

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...