Üniformalı Hizmetleri Üniversitesi intramural programı tarafından finanse edilmektedir.

1. Yenidoğan Rat Beyin Dokusu diseksiyonu <ol><li> Chill Leibovitz L-15 şartlandırılmış ortam (Leibowitz L-15 +% 0.1 BSA, 1 +% Pen / Strep) ile 4 ° C. Sıcak kültür ortamına (DMEM% 10 FBS + +% 1 penisilin / streptomisin) 37 ° C&#39;ye kadar Buz üzerinde klimalı L-15 medya 4-5 ml 4 60x15 mm Petri kapları hazırlayın. % 100 etanol ile tüm cerrahi aletleri sterilize edin. </li><li> % 70 etanol ile P2 yenidoğan rap yavrular durulayın. (P1-P5 arası Yenidoğan sıçan yavrularının bu protokolü kullanılabilir.) </li><li> Hızla steril keskin bir makas ile yavru sıçan başını kesmek ve% 70 etanol içine hemen kafasını bırakın. 5 sıçan yavrularının toplam tekrarlayın sonra tuzlu su çözeltisi içine başlarını aktarmak. </li><li> Buz üzerinde 5 ml L-15 solüsyonu (Leibowitz L-15 +% 0,1 BSA +% 1 Kalem / Strep) - 4 Petri içine başkanları ve yerden tüm beyinleri çıkarın. </li><li> 1.4 çöp kalan yavrular için - prosedürleri 1.2 tekrarlayın. </li><li> Beyin meninks çıkarın ve des aktarmakL-15 solüsyon 4-5 ml ile yeni bir petri içine ired beyin dokusu (yani, korteks, serebellum, tüm beyin, vb) (Leibowitz L-15 +% 0,1 BSA +% 1 Kalem / Strep) buz üzerinde. </li></ol> 2. Karışık Glial Hücre Nüfus hazırlanması <ol><li> Makas veya bıçakla zarar veren beyin dokusu olmaksızın, steril bir 50 ml konik tüp için 10 ml pipet ile doku aktarımı. </li><li> 4 ° C sıcaklıkta 5 dk için 2500 RCF azından konik santrifüje </li><li> Süpernatant aspire. Taze L-15 medya 4-5 ml ekle (Leibowitz L-15 +% 0,1 BSA +% 1 Kalem / Strep). </li><li> Steril bir 10 ml pipetle Pipet doku yukarı ve aşağı 10 kez. </li><li> Taze bir 50 ml konik boru üzerine bir hücre süzgecinden (100 mikron gözenek) yerleştirin. 5 ml steril bir pipetle ve hücre süzgecinden boşaltım pipet ile bir kez pipetle doku yukarı ve aşağı doğru, konik tüpe hücre süzgecinden malzemenin dağıtım. </li><li> Taze L-15 ortam 4-5 ml (sahip bir hücre süzgecinden durulama Leibowitz L-15 +% 0.1 BSA +% 1 Pen / Strep). </li><li> 4, 5 dakika boyunca 2500 RCF azından süzme hücreleri ile konik santrifüje ° C. </li></ol> 3. Karışık Glial Hücre Kültürleri Kaplama ve Bakım <ol><li> Her sıçan beyni yavru işleme için, kültür ortamı, 12 ml ilave edilerek 1 steril bir T-75 şişeye hazırlanması (DMEM% 10 FBS + +% 1 penisilin / streptomisin) her bir balona. </li><li> Pelet hücrelerden süpernatan aspire ve hücre pelletini kültür medya 5-6 ml ekleyin. 10 ml lik bir pipet yardımıyla 10 kez aşağı yukarı Pipeti ve. </li><li> Her bir T-75 balona hücre süspansiyonu eşit hacimde aktarın. </li><li> 1-3 hafta olmak üzere toplam 37 ° C&#39;de% 5 CO 2 inkübatörde matara inkübe edin. </li><li> Flasks ilk 5 gün süre ile inkübe, ve beşinci gününde, taze ortam 12 ml ile, her şişe içinde kültür ortamı değiştirmek ve inkübatöre geri edilmelidir. Daha sonra, her 3 gün, birleştiği elde etmek için taze ortam 12 ml ile, her şişe içinde şartlandırılmış ortam yerini alır. Bu yapmanız gerekmektedirne çok dikkatli hücrelerinin tutunup matara alt dokunmadan. </li></ol> 4. İlköğretim mikroglia İzolasyon ve Kaplama <ol><li> Mikst glial kültürler tamamen konfluent olduktan sonra, inkübatör gelen şişeler kaldırmak ve çevresel hava ile gaz değişimi önlemek için parafilm ile şişesi kapakları kapsamaktadır. </li><li> 37 1 saat için 100 rps (Lab Companion SI-600, Jeio Tech) de matara çalkalayın ° C </li><li> Şişeye yüzeyinde astrosit tabakası bozmadan 10 ml pipetle her şişe ortam Collect. 50 ml konik tüpler ortam yerleştirin. Inkübatöre matara ve dönüş matara taze Medya ekle. </li><li> 4, 5 dakika boyunca 2500 RCF azından konik tüpler santrifüje ° C. </li><li> Tüm konik tüpler süpernatanıyla aspire. Hücre peletler, yüksek saflıkta mikroglia hücrelerdir. Mikroglia kaplama ortam (DMEM +% 10 FES +% 1 penisilin / streptomisin) 1 ml Pastör pipetiyle peletleri. </li><li> Kullanılarak tekrar süspanse edilir ortam içinde hücre yoğunluğu saymaBir hemositometre. </li><li> Ml yoğunluğa başına 2 x 10e5 hücreler elde etmek için mikroglia kaplama ortam uygun bir hacmi ekleyin. Deneysel analiz için uygun Plate. </li><li> Mikroglia gecede eklemek için izin ver. </li></ol> 5.. Temsilcisi Sonuçlar Protokolü yüksek saflıkta ilköğretim mikroglia kültürlerde sonuçları yukarıda anlatılan. Gibi bir makrofaj / mikroglia hücre spesifik bir işaretleyici (Iba1) immünohistokimyasal olarak belirlenen, kaplama mikroglia kültürleri&gt;% 90 saf (Şekil 2). Ayrıca, astrosit, oligodendrosit ve nöron hücresi spesifik marker için boyama minimal kontaminasyon (Şekil 2) göstermektedir. Karışık glial hücre kültürleri kurduktan sonra, arka arkaya mikrogliya 4 kat kadar çalkalama ile izole edilebilir. İlk mikroglia izolasyonu yaklaşık 2.2 x 106 hücre / ml veya 10 matara ve beklenen verimi yaklaşık 9 milyon hücre birbirini izleyen sha azalır verecektirke. Bu yöntem ile izole mikroglia fagositoz yeteneği, nitrik oksit üretimi gibi işleyen ve nöronal toksisite (Şekil 3) 13-14 araştırmak için başarıyla kullanılmaktadır. <img alt="Şekil 1" src="/files/ftp_upload/3814/3814fig1.jpg" /> Şekil 1. Karışık glial hücre kültürleri ve primer mikroglianın izolasyonu hazırlanması için protokol bakış. <img alt="Şekil 2" src="/files/ftp_upload/3814/3814fig2.jpg" /> Şekil 2. Olarak astrosit mikroglia (Iba-1, kırmızı), (GFAP, yeşil), nöron (Neun, kırmızı) ve oligodendrosit kullanılarak immünohistokimyasal analizi ile doğrulanmış yüksek saflıkta mikroglia İzolasyon (CC1, kırmızı) özgül belirteçler. DAPI (mavi) tarafından kültürdeki hücre DNA lekelenmesi de gösterilir. <img alt="Şekil 3" src="/files/ftp_upload/3814/3814fig3.jpg" /> Şekil 3. Mikroglia yöntemi tarif izoleyatağı da dahil olmak üzere bir dizi deneyler, in kullanılabilir, fakat deneyler fagositozunu, fonksiyon deneyleri ve nörotoksik çalışmaları, bunlarla sınırlı olmamak edilmiştir. Bu çalışmalarda, biz (A) veya LPS (B) tedavi medya, ölçülebilir floresan ile işaretlenmiş lateks boncuk (kırmızı), (onların fagositoz artırmak mikroglia (Iba-1 pozitif, yeşil), kontrol maruz kaldığında bulduk C). Bundan başka, LPS (1ng/ml) tedavisi mikroglia (D), nitrik oksit üretimi arttırır. Son olarak, transwell insert ayrılmış veya doğrudan mikroglia-nöron iki kültür yoluyla, biz laktat dehidrogenaz (LDH) sürümü (E, F) ile ölçülen LPS ile inkübe mikroglia nöronal hücre ölümüne sebep bulduk.

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Çıkar çatışması ilan etti.

Bu protokol rutin araştırma deneyleri için saf ve sağlıklı mikroglia üretmek için kullanılır iken, prosedürün kritik parçalar sırasında teknik yönlerini dikkatle düşünülmesi izole mikroglia değişkenliği en aza indirecektir. İlk olarak, zamanında çalışan yenidoğan sıçanların yavrularının beyin dokusu, diseksiyon sırasında doku hipoksik ve iskemik hasarı en aza indirmek için gereklidir. Ancak, meninksler varlığı bu kültür şartlarında hızlı çoğalma hızı nedeniyle önemli f...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> 60 mm x 15 mm petri </td><td> Fisher marka </td><td> 0875713A </td><td></td></tr><tr><td> Keskin diseksiyon makas </td><td> Güzel Bilim Araçları </td><td> 14094-11 </td><td></td></tr><tr><td> Dumont # kavisli 7b forseps standart ip uçları, 11cm </td><td> Güzel Bilim Araçları </td><td> 11270-20 </td><td></td></tr><tr><td> Dumont # 5 forseps standart ipuçları, düz, 11 cm </td><td> Güzel Bilim Araçları </td><td> 11251-10 </td><td></td></tr><tr><td> 50 ml konik bir santrifüj tüpünde </td><td> VWR </td><td> 89039-656 </td><td></td></tr><tr><td> 5 ml serolojik pipet </td><td> Grenier Bio Bir </td><td> 606180 </td><td></td></tr><tr><td> 10 ml serolojik pipet </td><td> Grenier Bio Bir </td><td> 607180 </td><td></td></tr><tr><td> 100 mikron steril naylon hücre süzgecinden </td><td> Şahin </td><td> 35-2360 </td><td></td></tr><tr><td> 75 cm 2 doku kültürü şişeleri </td><td> Corning </td><td> 430641 </td><td></td></tr><tr><td> Dulbecco ortamı minimum gerekli (DMEM) </td><td> Gibco (Invitrogen) </td><td> 31053-028 </td><td></td></tr><tr><td> Leibovitz L-15 orta </td><td> Gibco (Invitrogen) </td><td> 11415064 </td><td></td></tr><tr><td> Fetal sığır serumu </td><td> Gibco (Invitrogen) </td><td> 16000-036 </td><td></td></tr><tr><td> Fetal at serumu </td><td> Balıkçı </td><td> SH3007402 </td><td></td></tr><tr><td> Penisilin-Streptomisin </td><td> Gibco (Invitrogen) </td><td> 15140163 </td><td></td></tr><tr><td> % 100 etanol </td><td> Warner Graham Şirketi </td><td> 64-17-5 </td><td></td></tr><tr><td> Fosfat 10X tuzlu su çözeltisi, pH 7.4 tamponlu </td><td> Kalite Biyolojik, inc. </td><td> 119-069-131 </td><td> Steril distile su 1X </td></tr><tr><td> Biohazard çanta </td><td> VWR </td><td> 14220-028 </td><td></td></tr><tr><td> Haemocytometer </td><td> Hausser Bilimsel </td><td> 1492 </td><td></td></tr><tr><td> CellBIND yüzeyi ile 6-de hücre kültür plakaları </td><td> Corning </td><td> 3335 </td><td></td></tr></tbody></table>

primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue

Memeli beyninde toplam hücresel nüfusunun yaklaşık% 12 için mikroglia hesabı. Nöronlar ve astrositler sinir sisteminin önemli hücre tipleri kabul edilirken, mikroglia enkaz ve patojenler için doku izlenmesi ve fagositik aktivite 1,2 ile parankiminde homeostazı koruyarak normal beyin fizyolojisi önemli bir rol oynamaktadır. Mikroglia nörodejeneratif hastalıklar, travmatik beyin hasarı ve sinir sistemi enfeksiyonu 3 de dahil olmak üzere yaralanma ve hastalık, bir dizi sırasında aktive edilir. Bu aktive edici koşullar altında, mikroglia, bunların fagositik aktiviteyi artırır morpohological ve proliferatif değişikliğe uğrar ve aktif reaktif oksijen ve nitrojen türlerinin, pro-inflamatuar kemokinler ve sitokinler salgılar, genellikle bir parakrin veya otokrin döngü 4-6 aktive. Bu mikroglial yanıtları nörolojik koşullarda hastalığı patogenezine katkıda gibi, araştırma m odaklandıicroglia garantilidir. Beynin hücresel heterojenite nedeniyle, in vivo deneyler sırasında yüksek saflıkta yeterli mikroglial örnek materyal elde etmek teknik olarak zordur. Mikroglia nöroprotektif ve nörotoksik fonksiyonları üzerine Mevcut araştırma tutarlı çalışması için yeterli verimi ile saf ve sağlıklı mikroglia üretmek için rutin bir teknik yöntem gerektirir. Biz, metin ve video, aşağı çeşitli uygulamalar için karışık glia kültürlerden saf primer mikroglia izole etmek için bir protokol mevcut. Kısaca, bu tekniğin karışık glial hücre kültürü üretmek için yenidoğan sıçanların yavrularının ayrışmış beyin dokusu kullanır. Karışık glial kültürler konfluent ulaştıktan sonra, primer mikroglia mekanik çalkalama kısa bir süre ile kültür izole edilir. Mikroglia sonra deneysel çalışma için yüksek saflıkta kaplanıyor. Bu yöntemin prensibi ve protokol olduLiteratürde 7,8 tarif. Buna ek olarak, birincil mikrogliya izole etmek için alternatif bir yöntem de 9-12 tarif edilmektedir. Homojenize beyin doku primer mikroglia 12 verim yoğunluk gradyanı santrifüjleme ile ayrılabilir. Ancak, orta boy santrifüjleme (45 dakika) ve hücre hasar ve aktivasyonu, aynı zamanda, zenginleştirilmiş mikroglia ve diğer hücre popülasyonları neden neden olabilir. Başka bir protokol kültürünün çalkalama 9-11 iken (16 saat) uzamış organizmalar çeşitli primer mikroglia izole etmek için kullanılmıştır. Çalkalandıktan sonra, süpernatan ortam mikroglia izole etmek için santrifüje tabi tutulur. Bu işlem iki aşamalı bir yöntem ayrıca izolasyonu mikrogliyal fonksiyonu ve aktivasyon karıştırmayı olabilir. Biz esas nedenlerle bir dizi için laboratuvar aşağıdaki mikroglia izolasyon protokolü kullanır: (1) primer mikroglia (2, daha sadık ölümsüzleştirdi kemirgen mikroglia hücre hatları daha vivo biyoloji simüle) Nominal mekanik bozulma potansiyeli hücresel disfonksiyon veya aktivasyon en aza indirir, ve (3) yeterli verim karışık glial hücre kültürlerinin geçiş olmadan elde edilebilir. Bu protokol mikroglia yalıtmak için yenidoğan sıçanların yavrularının beyin dokusu kullanır ve mikroglia önemli ölçüde izole mikroglia verimi, aktivasyon durumu ve fonksiyonel özelliklerini etkileyebilir izole etmek için büyük sıçan kullanarak bu dikkat etmek önemlidir. Yaşlanma mikroglia disfonksiyon, artmış nöro-inflamasyonu ve nörodejeneratif patolojileri ile bağlantılı olduğuna dair kanıtlar vardır, bu nedenle önceki çalışmalarda yaşlanmanın önemli bir parametre olduğu daha iyi nörodejeneratif hastalıklarda mikroglia rolünü anlamak için ex vivo yetişkin mikroglia kullandık. Bununla birlikte, ex vivo mikrogliya uzun süreler için kültür tutulması mümkün değildir. Bu protokolün bir kültürü primer mikroglianın ömrünü uzatır iken, bu nedenle, bu mikrogliya d davranırlar not edilmelidirin vivo ortamda bir tercüme ettiğimizde ifferently yetişkin mikroglia gelen ve in vitro çalışmalar dikkatle düşünülmelidir.

Watch this Scientific Journal Video about Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue at JoVE.com

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Beynin hücresel heterojenite birincil mikroglia Yalıtımlı fizyolojik ve patolojik hem de kendi rolünü araştırmak için esastır. Bu protokol bir mekanik izolasyon ve yüksek verim ve yüksek saflıkta sağlar karışık hücre kültürü tekniği için geçerli birincil mikroglia hücreleri açıklar<em&gt; In vitro</em&gt; Çalışma ve aşağı uygulamaları.

Yenidoğan Rat Beyin Dokusu Karışık Glial Hücre Kültürleri Primer mikroglia İzolasyonu

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

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JoVE Journal

Immunology and Infection

39.1K Görüntüleme.  Uniformed Services University. Beynin hücresel heterojenite birincil mikroglia Yalıtımlı fizyolojik ve patolojik hem de kendi rolünü araştırmak için esastır. Bu protokol bir mekanik izolasyon ve yüksek verim ve yüksek saflıkta sağlar karışık hücre kültürü tekniği için geçerli birincil mikroglia hücreleri açıklar In vitro Çalışma ve aşağı uygulamaları.

Video: Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Yetişkin Karaciğer Hücrelerinin Karma İlköğretim Kültürler Makrofajlar yalıtın Basit ve Etkin Yöntem

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

İmmünoloji ve Enfeksiyon

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

İlköğretim İnsan Keratinositler gelen Organotipik Radye Kültürlerin Üretimi

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Yenidoğan ve Erişkin Merkezi sinir sisteminin Kültüre Mikroglia

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Kemirgen Yenidoğan itibaren Korunan immunfenotipine ve İşlevselliği ile Kortikal mikrogliya İzolasyonu

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

Sıçan Testislerindeki Sertoli Hücreleri İzolasyon ve peritübüler Hücreler

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

İskemik Fare Beyin gelen Bağışıklık Hücreleri İzolasyon ve Akım Sitometrisi Analizi

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

Fare Primer Hepatosit Kültürler bir inflamatuar yanıtın indüksiyon

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

Hücresel stres ve ayrık oksitosin salgılayan beyin çekirdekleri içinde yenidoğan sıçan iltihabı önce ve sonra ilk kolostrum besleme değerlendirilmesi

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

Protozoa Parazit Enfeksiyonunda Primer Astrosit ve Mikroglianın Eşlik Etmesinin İzole Edilmesi

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...

Timus ve Kemik İliğinden Stromal Hücre İzolasyonunun Optimize Edilmesi

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Yazar Spotlight: Tek Hücre Dizilimi için Stromal Hücre İzolasyonu Kullanarak Hematopoietik Araştırmaları İlerletmek

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Hematopoetik Organlardan Stromal Hücre İzolasyonu

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...