Biz sitometrisi ile yardım için Marcelo Toro kabul etmek istiyorum. Bu çalışma, İsveç ve Kalp-Akciğer Vakfı, AB Komisyonu 7.ÇP &quot;CardioCell&quot;, İsveç Araştırma Konseyi, AFA sigortalar ve ALF tarafından desteklenmiştir. OB Deutsche Forschungsgemeinschaft tarafından desteklenmiştir.

1. Kardiyak Çekirdek İzolasyonu <ol><li> % 1 BSA / PBS kaplama solüsyonu 10 ml Coat ultrasantrifüjdeki tüpler (Beckman santrifüj tüpünde # 363.664). Tüpler Cap ve onları bir tüp rotator 30 dakika döndürün. Kaplama çözümü çıkarın ve santrifüj tüplerine hava kuru (fare kalp başına bir tüp farklı canlı türlerinin tek bir fare kalpleri, dönüşümlü olarak 5&#39;e kadar fare kalpleri veya kalp dokusunun 1 gr analizi için gerekli izin (örneğin insan) işlenebilir bir tüp içinde). </li><li> Bütün aşağıdaki adımları buz üzerinde yapılmalıdır. Bistüri ile taze veya ek donmuş fare kalpten sol ventrikül parçalara ayır. Not, bu protokol fare kalp için optimize edilmiş değil, aynı zamanda sıçan veya insan kalp adapte edilebilir. Alternatif olarak, farklı canlı türlerinin kalp dokusunun 1 g kadar kullanabilirsiniz. </li><li> Bistüri ile küçük hücreler halinde numune kesin. </li><li> Lizis tamponu ile 15 ml ile doldurulmuş bir 50 ml Falcon tüp içine doku parçaları transfer edin. </li><li> Homojenize10 sn için 24,000 rpm bir T-25 Ultra-Turrax prob homojenizör (IKA) ile kalp dokusu. </li><li> 30 ml lizis tamponu eşit hacmi Homojenat sulandırınız. </li><li> Daha fazla doku ve serbest çekirdekler homojenize bir cam douncer (40 ml) kullanın. Büyük bir boşluk havaneli sekiz vuruş yapın. </li><li> Ham çekirdek, 100 mikron ile 70 mikron naylon örgü hücre süzgecinden (BD Biosciences), ardışık yoluyla izole geçirin. </li><li> Ham çekirdekler 10 dakika süreyle 700 xg&#39;de bir soğutmalı santrifüj (4 ° C) izole aşağı Spin. </li><li> Ters tüpler tarafından dikkatle Süpernatantı ve kağıt havluyla tüpün içini silin. Çekirdekler pelet rahatsız dikkat edin. </li><li> Ham çekirdek çözümü birkaç kez pipetleme ve aşağı sakaroz tampon 5ml izole eritin. Çözünmüş pelet ile sükroz tampon, bir başka 25 ml ilave edilir. </li><li> Kaplamalı ultrasantrifüjdeki tüp (adım 1.1) için taze hazırlanmış sakaroz tampon 10 ml ekleyin. &lt;/ Li&gt; <li> Dikkatli bir şekilde adım 1.9 den sükroz tampon içinde yeniden süspanse çözünmüş çekirdekler pelet ile sükroz tampon katma 10 ml kaplamak. </li><li> Bir JS13.1 ücretsiz sallanan rotor koymadan önce santrifüj tüplerine dengelemek ve yüksek hızlı santrifüj (Beckman Avanti S-25) içine rotor yerleştirin. </li><li> 60 dakika süre ile 4 ° C&#39;de 13,000 x g&#39;de çekirdekler Örnek dönerler. </li><li> Spin tamamlandığında, rotor dikkatlice tüpleri çıkarın ve ters tüpleri ile süpernatantı atın ve kağıt havlu ile tüplerin içerisinden kalan enkaz silerek. </li><li> Çekirdeklerin depolama tamponu (NSB artı tampon) 1 ml içinde çekirdekleri pelet eritin. Not: NSB artı bir DNA stabilizatörü olarak 1.5 mM spermin içerir. </li><li> Kardiyomiyosit çekirdeklerinin İmmünoboyama, adım 2.1 ile devam edin. </li></ol> 2. Akım Sitometri için pozitif <ol><li> Immünboyama için negatif kontrol hazırlayın. Bir çekirdek numunenin 20 ul dışında tablet ve bir atınNSB artı tamponu dd 980 ul. </li><li> Immunolabel kardiyomiyosit çekirdeklere 1:500 dilüsyon içinde çekirdek örnek anti-pericentriolar malzeme 1 antikoru (tavşan anti-PCM-1, Atlas Antikorlar) ekleyin. Adım 2,1 hazırlanan negatif kontrol etmek anti-PCM-1 antikoru ile aynı seyreltme, in izotip antikor ekleyin. </li><li> 4 ° C&#39;da negatif kontrol ve numune inkübe edin. </li><li> NSB artı tamponu (10 dakika için 700 xg&#39;de bir soğutmalı santrifüj (4 ° C) tüpler aşağı döndürün. Süpernatan atın ve NSB artı tampon 1 ml çekirdekleri pelet erimesi) ile en az bir kez negatif kontrol ve numune yıkayın. </li><li> 1:1000 dilüsyon negatif kontrol ve numune tüpüne anti-tavşan floresan sekonder antikor (FITC veya APC) ekleyin. </li><li> 1 saat boyunca 4 ° C de negatif kontrol ve numune inkübe. </li><li> NSB artı tamponu (10 dakika için 700 xg&#39;de bir soğutmalı santrifüj (4 ° C) tüpler aşağı spin ile en az bir kez negatif kontrol ve numune yıkayın. Süpernatant atın ve) NSB artı tampon 1 ml çekirdekleri pelet çözülür. </li><li> Akım sitometri analizi ve sıralama ile devam edin. </li></ol> 3. Akım Sitometri <ol><li> Adım 1.1 &#39;de tarif edildiği gibi sıralama akış sitometrisi başlamadan önce,% 1 BSA / PBS çözeltisi ile Coat çekirdekler toplama tüpleri (Falcon 15 ml). </li><li> Örnek 30 um ve bir hücre süzgecinden negatif kontrol filtre ve (BD Akın) ilk akış sitometresi ile negatif kontrol yerleştirin. Forward scatter (FSC), forward scatter darbe genişliği (FS darbe genişliği) ve yan dağılım (SSC) (Şekil 2a ve b) dayalı, çekirdekler ve mayoları (tek çekirdek) tanımlamak için birinci ve ikinci kapısı tanımlayın. Örnek leke bir DNA (DRAQ5 (1:500)) ekleme başlangıçta çekirdek nüfusu belirlemek için yardımcı olabilir. </li><li> Immunolabeled örnek yükleyin ve non-kardiyomiyosit çekirdek (PCM-1-negatif) gelen kardiyomiyosit çekirdeklerin (PCM-1-positve) izole etmek için üçüncü kapısı tanımlar. Sıralama başlayın(Şekil 2 c ve d). </li></ol> İsteğe bağlı: nükleer DNA içeriği (ploidi) analiz etmek ve çekirdekleri (örn. Hoechst 33342 veya DRAQ5) (Şekil 2e) için leke uygun bir DNA eklemek hücre döngüsü analizi gerçekleştirmek için. <ol start="4"><li> Flow sitometri sıralama sonra, buz çekirdekleri yerleştirin ve sıralama saflık (Şekil 3a ve b) belirlemek için yeniden analiz. </li><li> 15 dakika için soğutmalı santrifüj 1500 xg&#39;de toplama tüpleri içinde sıralanır çekirdekleri aşağıya Spin. </li><li> Aşağı uygulama ile uyumlu bir tampon içinde çekirdekler pelet içinde çözülür. </li></ol> 4. Temsilcisi Sonuçlar Çekirdek morfoloji ve DNA bütünlüğü lekeleri tarafından değerlendirilmeli ve mikroskobu (Şekil 1) ile görüntülenebilir. Başarılı PCM-1 etiketleme Epifloresans mikroskop ile ve akım sitometri (Şekil 1 ve Şekil. 2c ve d) tarafından değerlendirilebilir. PCM-1-positiettik ve negatif popülasyonunda (Şekil 2c ve d) birbirinden ayrılmalıdır. Fare sol ventrikül tüm çekirdeklerin yaklaşık% 30 (Şekil 2d) kardiyomiyosit çekirdeklerinin olmalıdır. Saflık Sıralama sıralanır çekirdeklerin (Şekil 3a ve b) yeniden analiz edilerek değerlendirilebilir. Her iki çekirdek popülasyonları% 95 aşan bir sıralama saflıkta olmalıdır. <img alt="Şekil 1" src="/files/ftp_upload/4205/4205fig1.jpg" /> Şekil 1.. PCM-1 kardiyomiyosit çekirdekler tanımlar. Kardiyak çekirdekler (a) PCM-1 (b) ve yetişkin bir fare kalbinde Nkx2.5 (c) antikorları ile boyanmış edilir. (D) PCM-1-etiketli çekirdekleri kardiyomiyosit sitoplazma (miyozin ağır zincir (MHC)) ile çevrilidir ve PCM-1 boyanmasının (ölçek çubukları 20 mikron ve 10 kardiyomiyosit çekirdeklerinin doğru kimlik belgeleyen, transkripsiyon faktörü Nkx2.5 ifade edilir mikron (d, inset)). (E) DNA ile görselleştirildiği Kardiyak çekirdekleri izole DRAQ5 leke. (F ve g) CardiomyocYTE çekirdekler PCM-1 (ölçek çubuğu 10 mikron) karşı antikorlar ile etiketlenir. Not, doku bölümünde miyosit çekirdek ve izole çekirdek (oklar), PCM-1 epinuclear boyanma paterni. <img alt="Şekil 2" src="/files/ftp_upload/4205/4205fig2.jpg" /> Şekil 2. Kardiyomiyosit çekirdeklerinin Akış sitometrik sıralama. (A) Kardiyak çekirdeklerin forward scatter (FSC) ve yan dağılım (SSC) ile tanımlanır. (B) ikinci bir kapısı FSC ve FS darbe genişliği 5 ile tek çekirdek tanımlar. (C, d) Fluorescent perdeleme kalp dokudan kardiyomiyosit çekirdeklerin ayrılması (PCM-1-pozitif) ve non-kardiyomiyosit (PCM-1-negatif) çekirdekler sağlar. (E) Fare kardiyomiyosit çoğunlukla (&gt;% 80) diploit (2n) olan, sadece küçük bir alt kümesini (4n) 6 tetraploid olduğunu. Not, insan kardiyomiyositlerin poliploidi çekirdeğinin daha yüksek bir frekans (&gt; 2n) 7,8 içerir. <img alt="Şekil 3" src="/files/ftp_upload/4205/4205fig3.jpg" /> <strong&gt; Şekil 3. Sıralanır kardiyomiyosit ve non-kardiyomiyosit çekirdeklerin saflığı analizi. Sıralanır non-kardiyomiyosit (a) ve kardiyomiyosit çekirdekler (b) yeniden analizi. Hem nüfusları% 99 aşan bir sıralama saflık göstermektedir.

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Çıkar çatışması ilan etti.

Kardiyomiyosit çekirdeklerinin doğru tespit miyokard 2,3 rejeneratif süreçlerinin analizi için çok önemlidir. Taze dokudan kardiyomiyositler izole etmek için, geleneksel teknikler özellikle hücre dışı matris proteinlerine enzimatik sindirimi ve düşük hızlı santrifüjleme ile interstisyel hücreleri daha sonra saflaştırma dayanmaktadır. Embriyonik kök hücreler (ESC) ile yaşayan kardiyomiyositlerin saflaştırma gibi Sirpa 9 veya mitokondriyal boyalar 10 gibi yüzey...

<table border="1"><tbody><tr><td> 1. Lizis Tampon </td></tr><tr><td> Reaktif Adı </td></tr><tr><td> 0,32 M sakaroz </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM CaCl2 </td></tr><tr><td> 5 mM magnezyum asetat </td></tr><tr><td> 2.0 mM EDTA </td></tr><tr><td> 0.5 mM EGTA </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 2. Sakkaroz tampon </td></tr><tr><td> Reaktif Adı </td></tr><tr><td> 2.1 M sakkaroz </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM magnezyum asetat </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Çekirdekler depolama tamponu (NSB plus) </td></tr><tr&gt; <td> Reaktif Adı </td></tr><tr><td> 0,44 M sakaroz </td></tr><tr><td> 10 mM Tris-HCl (pH = 7,2) </td></tr><tr><td> 70 mM KCl </td></tr><tr><td> 10 mM MgCl2 </td></tr><tr><td> 1,5 mM spermin </td></tr></tbody></table><table border="1"><tbody><tr><td> Reaktifler ve Ekipmanları </td><td> Şirket </td></tr><tr><td> Izotip tavşan IgG-ChIP Grade # ab37415 </td><td> Abcam </td></tr><tr><td> Tavşan anti-PCM-1 antikoru, # HPA023374 </td><td> Atlas Antikorlar </td></tr><tr><td> Eşek sn. antikoru, anti-tavşan Alexa 488 Fluor, # A-21206 veya eşdeğer san. floresan antikor </td><td> Yaşam Teknolojileri </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> Hücre süzgeçler 30 mikron, 70 mikron ve 100 mikron </td><td> BD Biosciences </td></tr><tr><td> Cam douncer (40 ml) ve&quot;L&quot; havaneli </td><td> VWR (Wheaton Sanayi A.Ş.) </td></tr><tr><td> T-25 Ultra-Turrax Homojenizatör </td><td> IKA Almanya </td></tr><tr><td> Aracı S25 N-18 G Dispergatör </td><td> IKA Almanya </td></tr><tr><td> Beckman Avanti Santrifüj </td><td> Beckman Coulter </td></tr><tr><td> Falcon tüpler 15 ml ve 50 ml </td><td> VWR </td></tr><tr><td> Beckman Santrifüj Tüpleri # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 ücretsiz sallanan rotor </td><td> Beckman Coulter </td></tr><tr><td> Akını sitometresi </td><td> Beckman Coulter </td></tr><tr><td> Tüp Rotator </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

En stratejileri sadece sitoplazmik protein işaretleyicileri 1 güveniyor gibi kardiyomiyosit çekirdeklerinin belirlenmesi doku kesitlerinde zor olmuştur. Gibi çoğalması ve apoptosis gibi kardiyak miyositler Nadir olaylar kardiyak miyosit çekirdeklerinin doğru bir tanımlama homeostazında ve patolojik durumların 2 hücresel yenilenme analiz gerektirir. Burada, pericentriolar malzeme 1 (PCM-1) ve sonraki akım sitometri sıralama karşı antikorlar ile yoğunluğu sedimantasyon ve immün tarafından otopsi doku kardiyomiyosit çekirdekleri izole etmek için bir yöntem sağlar. Bu strateji, taze doku ve dondurulmuş arşiv malzemesi gayet iyi çalışma avantajı ile yüksek verimlilik analizi ve izolasyon sağlar. Bu da zaten biyobankalar toplanan malzeme çalışma kolaylaştırır. Bu teknik uygulanabilir ve türler, geniş bir aralık içinde test edilmiştir ve karbon-14 3, hücre-CY olarak birden çok alt-uygulamalar için uygundurcle analizi 4, timidin analogları görselleştirme (örneğin BrdU ve DUK) 4, transcriptome ve epigenetik analizi.

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Kardiyak çekirdeklerin yoğunluğu sedimentasyon ile izole edilmiş ve akım sitometri kardiyomiyosit çekirdeklerinin belirlemek ve sıralamak için pericentriolar malzeme 1 (PCM-1) karşı antikorlar ile immunolabeled edilir.

Post-mortem Doku gelen kardiyomiyosit Çekirdeklerin İzolasyonu

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

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16.9K Görüntüleme.  University of Lund. Kardiyak çekirdeklerin yoğunluğu sedimentasyon ile izole edilmiş ve akım sitometri kardiyomiyosit çekirdeklerinin belirlemek ve sıralamak için pericentriolar malzeme 1 (PCM-1) karşı antikorlar ile immunolabeled edilir.

Video: Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Kısmen Pancreatectomized hastalar İnsan Islets izolasyonu

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

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Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

İnsan Prostat Kanseri Örneklerinden Kanser Kök Hücreleri İzolasyon

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

Yetişkin fareler atriyal Miyoksit izolasyonu

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

Optogenetik Modüle Kardiyomiyosit Aktivitesinin Elektromekanik Değerlendirilmesi

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

HiPSC Türetilmiş Kardiyomiyosit Kullanarak İskemik Kalp Hastalığı Modeli ve Kardiyomiyosit Kontraksiyonunun Video Tabanlı Karşılaştırılması

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

İnsan Viseral Yağ Dokusundan Canlı Adiposit ve Stromal Vasküler Fraksiyonun RNA Analizi ve Makrofaj Fenotiplemesine Uygun İzolasyonu

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

Miyokard Enfarktüslü Farelerde Ultrason Rehberliğinde İndüklenen Pluripotent Kök Hücre Kaynaklı Kardiyomiyosit İmplantasyonu

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

Miyokard Enfarktüsü Sonrası Yetişkin Fare Kalbinden Tek Hücreli RNA Dizilimi için Kardiyomiyosit Olmayan Hücre Süspansiyonunun Hazırlanması

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

İnsan Tedavisi için Yenilikçi Bir Ksenojenik İçermeyen Yöntemle İnsan Adipoz Türevi Kök Hücrelerin İzolasyonu ve Kültürü

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

İnsan Keloid Dokusundan Primer Dermal Fibroblastların İzolasyonu, Kültürü ve Karakterizasyonu

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...