Bu eserler Doğa Bilimleri ve Kanada&#39;nın Mühendislik Araştırma Konseyi (RGPIN 238382-2011) ve Sağlık Araştırması (MOP115076) Kanada Enstitüleri hibe yoluyla destek verdi. Heather Paul Doğa Bilimleri ve Kanada&#39;nın Mühendislik Araştırma Konseyi Lisansüstü Burs ve Alberta yeniliklerin Health Solutions burs tarafından desteklenmiştir. Megan Hallam Doğal Bilimler ve Mühendislik Araştırma Konseyi Lisansüstü Bursu, bir Frederick Banting ve Charles Best Kanada Yüksek Lisans Bursu ve bir Alberta Çocuk Hastanesi Araştırma Enstitüsü Eğitim Genetik Ödülü, Çocuk Gelişimi ve Sağlık tarafından desteklenmiştir.

Bu protokol Calgary Hayvan Bakım Komitesi Üniversitesi tarafından onaylanmış ve Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu conformed oldu. Offspring 1. Ayrı Barajı <ol><li> Önce 12 sağma 5 dakika, en az onun soyundan gelen baraj ayırın. NOT: Baraj ancak uzun 4 saat daha ayrılık dönemleri süt bileşimini 14 değiştirebilir, ayrılık 1,6,13 sonra 5-6 saat kadar Sağılan edilebilir. Ayırma zaman süt toplama hacmi 12 darbe görünmüyor olsa da, tutarlı bir ayırma zamanı çalışma boyunca muhafaza edilmesi tavsiye edilir. Laktasyon 15 boyunca değişebilir Süt bileşimi, bu nedenle girişimleri tutarlı süt toplama gününü tutmak için yapılmalıdır. Maksimum süt üretimi laktasyon günde 14 12 oluştuğu ileri sürülmektedir. </li><li> Bir ısınma odasını kullanarak, yavrular uygun vücut temperatu muhafaza edebiliyoruz sağlamaksağma prosedür süresince annelerinin olmaksızın yeniden. NOT: Aşağıda sunulan çalışmada, sağım nedenle hiçbir ısınma odası kullanıldı, barajlar yaklaşık 22 haftalık ve yavru 21 gün eski zaman, sütten gerçekleştirildi. </li></ol> 2. Set-up ve Hazırlık <ol><li> Sağım işlemi için gerekli olan tüm malzemeleri toplayın. NOT: Tüm malzemeler malzeme ve ekipman tabloda bulunabilir. </li><li> Sağım gerçekleşecek ve altı-pad emici tezgah pedi kapsayacak bankta bir ısıtma yastığı yerleştirin. </li><li> Anestezik sistemini ayarlayın. Sistemi yeterli oksijen ve başlamadan önce izofluran olduğundan emin olun. Makineye ilk anestezi indüksiyonu için kullanılacak anestezi maskesi takın. Yakınlarda ilk anestezik maske farklı ise anestezi bakımı için kullanılacak maske yerleştirin. </li><li> Oksitosin için 1 ml şırınga 25 G iğne takınaseptik teknik kullanılarak enjeksiyonu. </li><li> Maternal vücut sıcaklığı sağma işlemi sırasında muhafaza edilir, böylece, ısıtma yastığı açın. NOT: Çok ısınırlar ve yanıklara neden olmaz pedi sağlamak için ısıtma pedi sıcaklığını izleyin. Alternatif olarak, belirli bir sıcaklık için ayarlanabilir bir ısıtılmış cerrahi tablosu olarak, bir ısı kaynağı kullanır. </li></ol> 3. Baraj kullanma izofluran anestezisi <ol><li> Oksijen tankı açın ve min başına 1 L (1000 cc) akışını çevirin. Izofluran akışı açın ve% 5 olarak ayarlanmış. DİKKAT: anestetik doğrudan solumaktan kaçının ve anestezik buharların birikmesini önler. </li><li> Baraj anestezisi. </li><li> Gerekirse emici tezgah pad üzerinde baraj sırtüstü yerleştirerek, bakım maskesi üzerinden geçiş. Pedal refleksi eksikliği anesthetization onaylayın. </li><li> Anestezi idamesi için% 2-3 izofluran akışını azaltın. Sürekli de emin olmak için prosedür boyunca baraj izlemeksolunum pression oluşmaz. NOT: anestezi altında, barajın gözleri kuruma veya çizik olma gözleri önlemek için steril bir göz yağlama kullanılarak korunmalıdır kez. </li></ol> 4. Oksitosin Enjeksiyon <ol><li> Oksitosin emin olun (20 USP Birimleri / ml) son kullanma tarihi geçmedi. / Alkol temizleme pedi silin steril alkol ile oksitosin flakon dezenfekte edin. </li><li> Aseptik teknik kullanarak, enjektöre oksitosin 2 IU (0.1 ml) hazırlamak. Sağılan olacak her baraj için yeni bir iğne ve şırınga kullanın. NOT: Oksitosin dozlar genellikle 1 tek enjeksiyondan 5 IU 1,6,12,16 değişir. Alternatif olarak, 4 lU / kg vücut ağırlığı kadar bir doz 12 kullanılabilir. Zorluk sağma karşılaşılırsa 2 IU tek bir dozu günde bir kez tekrar edilebilir. </li><li> Intraperitoneal oksitosinin enjekte edilir. Bir açıda, baş doğru iğne işaret ile karın sağ alt kadranda içine iğne takın15-30 °, 0.5 cm derinliğindedir. </li><li> Enjeksiyondan önce negatif basınç sağlamak için pistonu geri çekin. Herhangi bir sıvı (kan, idrar, bağırsak içeriği gibi) enjektöre aspire edilir ise, iğneyi çıkarın ve yeni bir iğne ve şırınga ile enjeksiyon girişimi. Hiçbir sıvı oksitosinin enjekte ve biyolojik tehlikeli kabına hemen iğne ve şırınga atmak aspire edilirse. </li><li> Süt boşaltma uyarma oksitosin yaklaşık 5-15 dakika bekleyin. </li></ol> Sağım Siteleri 5. Hazırlık <ol><li> Süt tahsil edilecektir hangi siteleri / emzikleri seçin. Süt herhangi emzik 12 alınabilir. </li><li> Kürk numune toplama zorluk neden olabilir hafifçe nedeniyle süt fitilleme için, makasları ile Sağılan edilecek emzikleri etrafında kürk çıkarın. Nazik olun - emzikleri çevresindeki deri çok hassastır ve kuru olabilir ve gibi çizik ve gözyaşları duyarlıdır. </li><li> Meme i SterilizasyonGerekli değil, ancak kürk çıkarıldıktan sonra, isteğe bağlı olarak, ılık suyla emziği temizleyin. Birden fazla site süt toplama için gerekli olabilir, en az iki site hazırlayın. NOT: Süt analizi mikrobiyal profilleme içeriyorsa, emzik alan iyot 5 ile sterilizasyon gerekebilir. </li></ol> 6. Süt Toplama <ol><li> Yavaşça elle toplama süt kovma, emzik tabanını sıkmak. NOT: Süt analizi mikrobiyal profilleme içeriyorsa, sütün ilk birkaç damla atılmalıdır. </li><li> Kılcal tüp dolum, bir kılcal tüp içine süt damlacıkları toplayın. Daha büyük hacimli (örneğin, 50 ul) sürecini kolaylaştırmak uyum Kılcal borular. Not: süt toplama zorluğu ile karşılaşılırsa, oksitosin ikinci bir doz uygulanabilir. Bu doz 4 IU toplam geçmemesi tavsiye edilir. </li><li> Steril bir mikrosantrifüj tüp ile içine kılcal tüpten süt koyunmikrosantrifüj tüpü yan meme sütü çekmek için kullanılan tüpün bitiş ucunu da dokunarak - Süt, kılcal hareket aracılığı kılcal tüp üzerinden çekilmesini dikkate alınmalıdır. NOT: 1 ml şırınga bağlı 18 G iğne kullanılarak tüp içine çekilir değil süt &#39;söndür&#39;. </li><li> Sürekli ağrı veya solunum depresyonu belirtileri için baraj izlemek ve buna göre izofluran akışını ayarlamak. </li><li> Yeterli süt seçilen süt analizi için toplanan kadar bu bölümde tarif edildiği gibi süt toplamak için devam edin. Creamatocrit ve protein konsantrasyonu belirlenmesi için aşağıda tarif edilen, süt 0,25 ml toplar. NOT: Süt hayal kırıklığı yavaşlatır veya seçilen sitesi yeterince süt sınırdışı etmezse, farklı bir sağım sitesini kullanın. Hayvan başına süt, yaklaşık 2,5 ml en fazla 17 toplanabilir veya meme başına 0.5 ml&#39;ye kadar olabilir. Yazarlar o anestezi altında toplam süresi yaklaşık 45-60 dakika ile sınırlı tavsiyer sağım 45 dk. </li><li> Taze süt örneklerinden creamatocrit ölçümü için bir mikrohematokrit tüp içine süt toplamak ve kil dolgu ile tüpün ucunu mühür. Baraj kimliği ile mikrohematokrit tüp Etiket ve dik süt örneği saklayın. </li><li> Sağma tamamlandığında, izofluran ve oksijen akışını kapatın. Baraj maskeyi çıkarın ve uyanık kadar baraj izlemeye devam. Tamamen bilinçli değilse, baraj, yerine doğrudan kafes yatak üzerinde daha iyileşme döneminde bir emici ped tezgah üzerine konulmalıdır aspire veya kurtarma sırasında baraj gözleri kaşıma olmaktan yatak önlemek için tavsiye edilir. NOT: sternum recumbancy korumak için yeterli bilinci yerine kadar sahipsiz baraj bırakmayın. </li><li> Başka analizler gerekiyorsa, -80 ° C süt dondurma. Diğer süt, -20 ° C 18 &#39;deki 3 saatin kadar 4 ° C de veya 5 ay boyunca saklanabilir olduğunu ileri sürmüşlerdir. </li></ol> 7. Creamatocrit Ölçümü <ol><li> Süt creamatocrit (süt örneğinde krem yüzdesi) 19 hesaplayarak sütündeki yağ içeriği tahmin edin. NOT: insan sütü ile ölçümler taze veya dondurulmuş ya süt, ancak taze süt daha çok ilişkili olduğu, bir creamatocrit ölçümü için kullanılabilir olduğunu göstermiştir (r r = 0.90 = 0.92 karşı) lipit konsantrasyonuna 11. Çözülmüş süt creamatocrit küçük bir azalma ile ilişkili olarak creamatocrit ölçümleri için taze veya dondurulmuş süt kullanımı 11 değerleri, çalışma boyunca tutarlı olmalıdır. </li><li> Taze süt için, bir mikrohematokrit tüp içine emziğin süt örneği toplamak; En azından doldurun ¾ dolu (yaklaşık 15-20 ul). Alternatif olarak, iyi karıştırıldıktan sonra kılcal tüpe toplanan örnekten taze süt çizin. Kil dolgu ile sonuna mühür. </li><li> Ile, hematokrit spinere kapiller tüp yerleştirinmühürlü uç santrifüj sağlanması dışarıya bakacak dengelidir. </li><li> Hematokrit dönüşünü (13.700 xg&#39;de 120 sn) başlayın. NOT: Sıkma süresi veya hız kullanılan santrifüj modeline bağlı olarak değişebilir. </li><li> Spin tamamlandıktan sonra santrifüj tüpü çıkarın ve creamatocrit hesaplanması için ölçümler yapmak. Bir krem ​​tabakası ve net bir tabaka haline numunenin ayrımını dikkate alınız. </li><li> Ölçüm ve akışkanın boru içindeki toplam uzunluğu ve yağ (krem) uzunluğunu çap pergeli ya bir cetvel kullanarak katman kaydedin. Not: creamatocrit olarak hesaplanan süt numunesi 19 olan krem tabakasının yüzdesi olarak ifade edilir (krem tabakanın uzunluk / süt sütununun toplam uzunluğu) 100 (Şekil 1A) x. Şöyle creamatocrit ölçümünden yağ konsantrasyon ve enerji değerlerini hesapla: Yağ konsantrasyonunun (g / L) = (creamatocrit (%) - 0.59) /0.146 (Şekil 1B) 19; Enerji değeri (kcal / L) = 290 + (66,8 * creamatocrit (%) (Şekil 1C) 19. </li></ol> 8. Protein Konsantrasyonu Tayini <ol><li> Bir protein standardı olarak sığır serum albümini kullanılarak, örneğin, bir Lowry protein testi 6 gibi standart protein deneyi kullanılarak toplam süt protein konsantrasyonunu belirleyin. Not: Süt protein ölçümleri deneyde standart eğri içine düşmesi süt seyreltilmesi gerekli olabilir. </li></ol>

<ol>
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Yazarlar ifşa etmek Çıkar çatışması var. Tüm hayvan deneyleri CCAC onaylı protokoller çerçevesinde yapılmıştır.

Investigations into maternal milk components have increased as interest in early life development research rises. As the sole source of nutrition during the neonatal period, the bioactive compounds in milk are essential for ideal growth and development, especially in the context of intestinal and immune health3. The method presented here is a simple, non-invasive method of collecting milk from the lactating rat in amounts sufficient for downstream analysis, such as oligosaccharide profiling6. The me...

Süt yenidoğan memeliler, bebek büyüme ve gelişme 1,2 için enerji ve besin sağlamak için beslenme tek kaynağıdır. Süt esas hücrelerde, lipid ve protein 1 oluşur, aynı zamanda enzimler, karbohidratlar, hormonlar, antikorlar, büyüme faktörleri, sitokinler, eksozom, microvesicles ve küçük RNA&#39;ların bu dahil olmak üzere, yavruların erken yaşam gelişmesini ayarlayabilen biyolojik olarak aktif bileşiklerin bir bolluk içerir microRNA 1,2 olarak. Bebeklerin hastalığa 2 daha az hassas olan anne sütüyle beslenen kanıtlarla ile birleştiğinde bağışıklık yavruları ve bağırsak sağlığı 3 kuruluşunda anne sütünün temel rolü, yaşamın erken döneminde hastalık süreçleri ile ilişkili süt bileşenleri ve ilgili moleküler mekanizmaları tanımlamanın önemini vurgulamaktadır eylemlerinde. Gelişmekte olan sıçan çeşitli beslenme, fizyolojik etkisini araştırmak için popüler bir modeldir ve erken kimyasal müdahaleler-Hayat gelişim 4. Sıçan süt analizi, bu nedenle anne ve yavru sağlığı içine yeni bilgiler sağlayabilir. Güncel bilimsel gelişmeler artık sağlık ve hastalık ile ilgili özel süt bileşenlerinin etkileri derinlemesine araştırmalar için olanaklarının artırılması sağlamak. Örneğin, süt bakteriyel profillerin dizi bebek gut 5 erken intestinal kolonizasyonunda kendi rolünü açıklamıştır, süt oligosakaritlerinin kütle spektrometresi analizi anne diyet 6 ve salgılanan microRNA derin sekanslama yoluyla süt oligosakarit profillerini değiştirilmesi fikir sağladı Anne sütünün yağ globülleri gen transkripsiyonu, metabolizma ve bağışıklık fonksiyonu 7&#39;de olası rollerini vurgular. Sıçan modelleri anne çalışmalarda 8,9 kullanılan en popüler model organizmaların birini temsil etmektedir. Bir avantajı sadece approximat süren kısa gebelik ve doğum dönemleri olduğunuely 21 gün, her; Bu nedenle emzirme gebelik başlangıcından itibaren toplam süresi değerli veriler elde edilebilir hangi kısa bir dönemi temsil eder. Farelere kıyasla farelerin büyük boyutlu, süt toplama bağlamında, süt ve süt toplama kolaylığı hacmine göre belirgin bir avantaj sağlayabilir; fare süt üretimi, örneğin, daha fazla süt üreten 10 ağır farelerin toplam vücut ağırlığına bağlı gibi görünüyor. Burada, emziren sıçanların sütüne elle toplanması için genel bir açıklama sağlanır. Bu protokol, düşük ekipman gerektiren ucuz, invazif olmayan ve daha sonraki alt akış analizler için yeterli miktarda süt toplamak için kullanılabilir. Kısaca, baraj süt boşaltma oksitosin tarafından uyarılan, izofluran ile anestezi edilir ve süt süt el ekspresyonu yoluyla kılcal tüpler içine toplanır. Son olarak, süt iki temel bileşenleri olarak proteinler, kısa bir TANIMI şişman vecreamatocrit ölçümleri 11 ve standart bir protein deneyi kullanılarak toplam protein konsantrasyonu ölçümü kullanılarak süt yağı içeriği tahmin n sunulmuştur.

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		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Milking</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringes</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">309602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ul Microdispenser Capillary Tubes</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">21-169D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxytocin (20 USP Units/ml)</td>
			<td style="width:191px;">Bimeda-MTC</td>
			<td style="width:119px;">1OXY015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PPC Vet Isoflurane Inhalation Anesthetic, 250 ml</td>
			<td style="width:191px;">Fresenius Kabi</td>
			<td style="width:119px;">M60302</td>
			<td>Used on the order of a veterinarian</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sterile Alcohol Prep Pad</td>
			<td style="width:191px;">Dukal</td>
			<td style="width:119px;">853</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Absorbent Bench Underpad</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">82020-845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Maxi-Therm Hyper/Hypothermia Blanket</td>
			<td style="width:191px;">Cincinnati Sub-Zero</td>
			<td style="width:119px;">274</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Rodent Anesthesia Machine with Vaporizer</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;"></td>
			<td>Subject to individual laboratory needs</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:353px;">Animal Masks</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;">50100/50102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge Tubes</td>
			<td style="width:191px;">Axygen</td>
			<td style="width:119px;">MCT-060-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChroMini Professional Trimmer</td>
			<td style="width:191px;">Wahl</td>
			<td style="width:119px;">-</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Creamatocrit</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin SafeCrit Plastic Microhematocrit Tubes (Untreated)</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">22-274-914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Critoseal Capillary Tube Sealant Tray</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">470161-478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin CritSpin Microhematocrit Centrifuge</td>
			<td style="width:191px;">Beckman Coulter, Inc</td>
			<td style="width:119px;">X00-004999-001</td>
			<td></td>
		</tr>
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milk collection in the rat using capillary tubes and estimation of milk fat content by creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

Wistar baraj çıkışta tarif edildiği gibi süt toplandı, yüksek protein bir denetim tüketilen (5 AIN-93 g, n =) ki (ağırlığında yaklaşık 22 haftalık 350 ila 400 g) (% 40 kazein ağırlık / ağırlık, n = 5) ya da yüksek prebiyotik lif (% 21.6 wt / wt, 1: oligofruktoz ve inülin, n = 4: 1 oran), gebelik ve emzirme döneminde, diyet. Oksitosin dozu IU 2 oldu. Süt kılcal borular kullanılarak toplandı ve daha sonra bir boru göre yağ konsantrasyonu ve enerji değerini tahmin etmek için kullanıldı c...

Watch this Scientific Journal Video about Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit at JoVE.com

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Kılcal Tüpler ve Creamatocrit Süt Yağ İçeriğinin Tahmin kullanarak Rat Süt Toplama

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

milk-collection-rat-using-capillary-tubes-estimation-milk-fat-content

Research

JoVE Journal

Biology

12.2K Görüntüleme.  University of Calgary. Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Video: Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biyoloji

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Sıçan Portal Fibroblastlar izolasyonu ile<em&gt; Yerinde</em&gt; Karaciğer Perfüzyon

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

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

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

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

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

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

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

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk Collection Methods for Mice and Reeves&#039; Muntjac Deer

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Fareler ve Reeves &#39;munçağı Deer için Süt Toplama Yöntemleri

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

Toplanması ve analizi<em&gt; Arabidopsis</em&gt; Floem EDTA-kolaylaştırdı Yöntemi eksuda

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Yüksek İçerikli Mikroskopi Kullanılarak Hücresel Redoks Profillemesi

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Karaciğer ve Süt örneklerinde vahşi tipli ve sialik asitler tayini ve<em&gt; CMAH</em&gt; Knock-out Fare.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

Drosophila melanogaster larva yağ gövdesinde açlığa bağlı otofajinin analizi

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

Decellularizasyon Yoluyla İskelet Kası Yağ İnfiltrasyonunun Miktar Tayini

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Yazar Spotlight: İskelet Kası Yağ İnfiltrasyonunun Decellularizasyona Dayalı Miktar Tayini  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.