Name: the preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation
Uploaded: 2009-01-06T23:59:00.000+00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation at JoVE.com

Femur ve süresince tibialar itibaren Kemik İliği Hasat <ol><li> 12-haftalık fare (BALB / c farelerde kullanarak ancak herhangi bir fare suşu ile bu işlemi yapabilirsiniz) - 6 femur ve süresince tibialar kemik iliği hasat prosedürün ilk adımı. Başlamak için, CO 2 inhalasyon (gösterilmeyecektir) fare euthanize. Hasat femur ve her birinin arka ayakları süresince tibialar. Sonra, bir tıraş bıçağı ile, kalça ve diz eklemleri kaldırmak için her iki ucunda femur kesmek ve kemik iliği maruz. </li><li> Benzer bir şekilde, diz ve ayak bileği komşu bölgeleri kaldırmak ve süresince tibialar iliği açığa çıkarmak için her iki ucundaki kesti. </li><li> 26-gauge iğne ile 3 ml şırınga alın ve RPMI takviye ile% 10 FBS, penisilin, streptomisin ve beta-mercaptoethanol (BME = 100 um) ile doldurun. Cımbız kullanarak, kemik RPMI medya içeren Petri üzerinde tutun. Şırınga iğnesi kemiğin bir ucunu takın ve kemik iliği dışarı atılması için piston bastırın. Şırınga iğnesi artık iliği dışarı floş kemik içinde yukarı ve aşağı hareket ettirilebilir. </li><li> Tüm kemikler için prosedürü tekrarlayın ve sonra kültürler kurulması ile devam. </li></ol> Kültürler kurulması <ol><li> Kemik iliği kültürleri kurmak için, bir hücre süspansiyonu doku kırmak için birkaç kez aşağı kızarmış kemik iliği pipet ve. </li><li> 50 ml konik tüpün üstüne bir 70 mikron filtre koyun ve hücre süspansiyonu filtre ekleyin. </li><li> Petri kabı RPMI ile bir kez yıkayın, sonra 70 mikron filtre durulama ekleyin. </li><li> 1 ml şırınga pistonu kauçuk ucunu kullanarak, 70 mikron filtre üstünde kalan kemik iliği adet eziyet. </li><li> RPMI ile filtre bir kez yıkayın. </li><li> Filtre yıkadıktan sonra, hücre süspansiyonu için 1200 rpm&#39;de 5 dakika santrifüj. </li><li> PBS içinde resuspending hücre pelletini yıkayın. </li><li> Hemasitometre kullanarak hücrelerin güvenin. Biz genellikle bir fare kemikleri (iki femurları artı iki süresince tibialar) ~ 90 milyon hücre edinin. Hücrelerinin sayımı sonra, onları tekrar 1200 rpm&#39;de 5 dakika santrifüj. </li><li> RPMI ve kısım son konsantrasyon ml başına 1 milyon hücre olduğunu yeterince RPMI içeren doku kültürü şişeleri içine küçük bir miktar hücrelerin tekrar </li><li> Ilgi hücre tipi kalkınmayı teşvik etmek sitokinler ekleyin. Bu durumda, sitokin interlökin-3 (IL-3 = 10 ng / ml), bazofil ve mast hücrelerinin gelişimini teşvik etmek eklenir. Bazofiller edinmek için, 10 gün boyunca kuluçkaya yatmaktadır. Mast hücreleri için, 5 hafta boyunca kuluçkaya yatmaktadır. Mast hücreleri oluştururken, medya ve IL-3 haftada bir kez değişmiş olmalıdır. </li><li> Burada mast hücreleri sadece kaplama sonra nasıl görünmelidir bir bakış. Bu görüntü, bazofil ve mast hücreleri interlökin-3 ilave edildikten sonra 10 gün mast iliği hücreleri farklılaşma göstermektedir. </li><li> İstenen hücre tipi bir kere elde edildiğinde, elektroporasyon adım ile devam edin. </li></ol> Electroporating Hücreler <ol><li> Elektroporasyon adım başlamak için, kültür şişelerinde hücrelerinin sayısını belirlemek. </li><li> Hücreler genellikle mL başına 10 milyon bir yoğunluk electroporated, bu yüzden konik tüpler için gerekli hücre sayısı transfer tüpler ve 1200 rpm&#39;de 5 dakika santrifüj. </li><li> Santrifüj sonra, medya aspirat, 1X PBS ile hücreleri yıkayın ve tekrar 1200 rpm&#39;de 5 dakika santrifüj. </li><li> PBS aspire ml başına 10 milyon hücre hücre süspansiyonu yapmak için Bio-Rad Gene Verici elektroporasyon tampon ekleyin. </li><li> Hücrelerin resuspending sonra, son bir konsantrasyonda ml başına 10 ila 20 mikrogram istenen plazmid DNA ekleyin. </li><li> 96-iyi elektroporasyon plaka üzerinde seçtiğiniz kuyu içine hücre süspansiyonu kısım 150 mikrolitre. </li><li> MXcell plaka odasında elektroporasyon plaka koyun ve kapağını kapatın. </li><li> Mast hücrelerinin transfeksiyon önce bir ön ayar veya saklanan bir protokol kullanarak sürece, elektroporasyon protokol MXcell içine programlanmış olmalıdır. Optimizasyonu deneyler sayesinde mast hücrelerinin yüksek transfeksiyon verimliliği kare dalga darbe protokolleri kullanarak ortaya keşfettiler. Biz 2000uF ve 1000 ohm 300V/20ms, 350/15ms, 350V/20ms ve 350V/10ms kare dalga darbeleri teslim plaka üzerinde elektroporasyon koşulları değişecektir. Protokol ve cihaz üzerine yüklendikten sonra, hücrelerin electroporate &quot;Darbe&quot; tuşuna basın. </li><li> Elektroporasyon tamamlandıktan sonra, bir doku kültürü plakası hücrelere aktarın. Biz genellikle interlökin-3 ml başına 10 nanogram 300 mikrolitre RPMI (% 10 FCS, penisilin, streptomisin ve beta-mercaptoethanol ile desteklenmiş) içeren bir 48-iyi doku kültürü plakası sıra her 150 mikrolitrelik elektroporasyon örnek aktarın. Hücreler 37 gece inkübe ° C, daha sonra ifade ve bastırılması için 24 saat sonra çalışılmalıdır. </li></ol> Transfection Sonuçlar Plazmid ml GFP başına 20 mikrogram kullanarak başarılı elektroporasyon sonra hücreler florasan mikroskobu görüntüsü, video gösterilir. Yaklaşık% 30 MXcell elektroporasyon sistem transfeksiyon verimliliği kullanılarak elde edilebilir. Sistem, hücre canlılığını korurken, transfeksiyon verimliliğini en üst düzeye çıkarmak için şartlarda değişiklik sağlar.

Daha genomik bilgi ortaya çıkıyor ve böyle bir siRNA olarak yeni araçlar geliştirilmiştir, fizyolojik ilgili hücrelerin kullanımı, hastalık yolları, protein-protein etkileşimleri, ve sinyal iletimi anlayışımızı ilerletmek için her zamankinden daha önemli hale gelmiştir. İlköğretim hücreleri, dokuları veya sıvıları doğrudan elde edilen ve in vitro olarak yetiştirilmektedir. Bu hücreler, genetik materyalin eksojen tanıtım yoluyla yolları da dahil, bir dizi manipüle edilebilir. Bu plazmid DNA veya birincil hücre içine siRNA tanıtmak için en etkili yolu elektroporasyon olduğunu giderek daha belirgin hale gelmektedir. Elektroporasyon geçici böylece ekzojen nükleik asitlerin hücre girmesine izin verir, hücre zarının geçirgenliğini artırmak amacıyla elektrik darbeleri hücreleri ortaya koyar. Transfeksiyon Bu yöntem, farklı hücre türleri / hatları ve, böylece mast hücrelerinin transfect kullanılabilir yanı sıra, diğer kemik iliği kaynaklı hücre arasındaki farklılıkların karşılamak için optimize edilebilir. Ayrıca, viral aracılı transfeksiyon aksine, elektroporasyon transfekte DNA boyutu sınırlar empoze, ne de kapsamlı bir hazırlık gerektirir değildir. Elektroporasyon lipid aracılı transfeksiyon aksine, daha az toksik olabilir ve transfekte nükleik asit endosomal yakalama sonuç vermez. Bio-Rad, bu hücrelerin MXcell elektroporasyon sistemi için özel olarak geliştirilmiştir. MXcell transfeksiyon verimliliği ve hücre canlılığı en üst düzeye çıkarmak için şartlarda değişiklik sağlar. Transfeksiyon verimliliği optimize etmek ve hücre ölümü, voltaj elektroporasyon parametreleri de dahil olmak üzere bir çok en aza indirmek için, kondansatör, direnç ve nabız uzunluğu ayarlanabilir ve değerlendirilmelidir.

<table><tbody><tr><td>Gene Pulser MXcell Electroporation System</td><td>Bio-Rad</td><td>165-2670</td><td></td></tr><tr><td>Gene Pulser Electroporation Buffer</td><td>Bio-Rad</td><td>165-2677</td><td></td></tr></tbody></table>

the preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation

Elektroporasyon birincil hücre içine plazmid DNA veya siRNA tanıtmak için en etkili yolu olduğu giderek daha belirgin hale gelmektedir. Gene Verici MXcell elektroporasyon sistemi ve Gen Verici elektroporasyon tampon, özellikle memeli hücrelerinde ve birincil ve kök cells.This video gibi zor-transfect hücreleri, nükleik asitlerin transfect geliştirilmiştir birincil fare kemik iliği hematopoetik hücre kültürleri nasıl kurulacağını gösteren ve sonra MXcell sistemi elektroporasyon için hazırlamak. Biz femur ve tibia izole ederek başlar. Femur ve tibia hem de kemik iliği biçilir ve kültürler kurulur. Kültüre kemik iliği hücreleri daha sonra transfekte ve analiz edilir.

Watch this Scientific Journal Video about The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation at JoVE.com

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

Bu prosedür, birincil fare kemik iliği hematopoetik hücre kültürleri kurmak ve transfeksiyon Gene Verici MXCell elektroporasyon sistemini kullanarak takip nasıl açıklamaktadır.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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25.7K Görüntüleme.  Bio-Rad Laboratories, Inc.  ChineseVersion 

Video: The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Fare ve insan hematopoetik progenitör hücre tarafından CRISPR/Cas9 yüksek verimli Gene bozulma

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Genetik

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

İzolasyon ve Kemirgen Kemik İliği intravenöz enjeksiyon Türetilmiş Monositler

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

İmmünoloji ve Enfeksiyon

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

Biyoloji

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Mürin Hematopoetik Kök Hücreler ve Lineage-kararlı progenitörlerin Fenotipik Analizi ve İzolasyon

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Hastalık Modellerinde Hematopoetik Hücrelerin İncelemi için Lentiviral CRISPR/Cas9-Aracılı Genom Düzenleme

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

Kemik İliği Kaynaklı Dendritik Hücrelerin Farelerden İzole Edilmesi ve Kültürlenmesi için Ekonomik ve Verimli Protokol

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

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 ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Kemik iliği türevi fare dendritik hücreleri ve makrofajlar floresan Fusion proteinlerin ifade

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

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Fare ve insan hematopoetik progenitör hücre tarafından CRISPR/Cas9 yüksek verimli Gene bozulma

İzolasyon ve Kemirgen Kemik İliği intravenöz enjeksiyon Türetilmiş Monositler

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Mürin Hematopoetik Kök Hücreler ve Lineage-kararlı progenitörlerin Fenotipik Analizi ve İzolasyon

Hastalık Modellerinde Hematopoetik Hücrelerin İncelemi için Lentiviral CRISPR/Cas9-Aracılı Genom Düzenleme

Kemik İliği Kaynaklı Dendritik Hücrelerin Farelerden İzole Edilmesi ve Kültürlenmesi için Ekonomik ve Verimli Protokol

Hematopoetik Organlardan Stromal Hücre İzolasyonu

Hematopoetik Organlardan Stromal Hücre İzolasyonu

Hematopoetik Organlardan Stromal Hücre İzolasyonu

Kemik iliği türevi fare dendritik hücreleri ve makrofajlar floresan Fusion proteinlerin ifade